PROBING THE BASE STACKING CONTRIBUTIONS DURING

TRANSLESION DNA SYNTHESIS

by

BABHO DEVADOSS

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Adviser: Dr. Irene Lee

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

January, 2009

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

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candidate for the ______degree *.

(signed)______(chair of the committee)

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*We also certify that written approval has been obtained for any proprietary material contained therein.

ii TABLE OF CONTENTS

Title Page…………………………………………………………………………………..i

Committee Sign-off Sheet………………………………………………………………...ii

Table of Contents…………………………………………………………………………iii

List of Tables…………………………………………………………………………….vii

List of Figures…………………………………………………………………….………ix

Acknowledgements…………………………………………………………….………..xiv

List of Abbreviations…………………………………………………………….………xv

Abstract………………………………………………………………………………….xix

CHAPTER 1 Introduction…………………………………………………………………1

1.1 The Chemistry and Biology of DNA…………………………………………2

1.2 DNA Synthesis……………………………………………………………….6

1.3 Structural Features of DNA Polymerases…………………………………...11

1.4 Models Accounting for Nucleotide Selectivity During DNA

Polymerization……………………………………………………………13

1.4.1 Active site tightness as a model for DNA polymerization……....13

1.4.2 Negative selection as model for nucleotide incorporation……….15

1.5 Translesion DNA Synthesis…..……………………………………………..17

1.6 Incorporation of Natural Nucleotides Opposite an Abasic Site……………..18

1.7 The Use of Non-Natural Nucleotides to Probe Shape Complementarity

During Translesion DNA Synthesis…………………………………..….....24

1.8 The Role of π-electron Surface Area During Translesion DNA Synthesis…26

1.9 An Alternate Model for DNA Polymerization………………………..…….29

iii 1.10 Statement of Purpose…...…………………………………………………..31

CHAPTER 2 Evaluating the Base Stacking Contributions of Alkylated and

Un-Natural Purine Nucleotides During the Translesion DNA

Synthesis Catalyzed by Bacteriophage T4 DNA Polymerase……….…………...33

2.1 Introduction…………………………………………………………………...34

2.2 Materials and Methods………………………………………………………..37

2.2.1 Purification of gp43 DNA polymerase and DNA substrates………….37

2.2.2 Modified purine nucleotides………………………...………………...37

2.2.3 Radiolabeling 5’ends of DNA substrates……………………………...38

2.2.4 Determination of kinetic rate and dissociation constants for dXTP

incorporation opposite an abasic site……….…………………..…..….39

2.2.5 Calculations of biophysical features of modified nucleotides..……..…41

2.2.6 Extension beyond an sbasic site……………….….……………………41

2.3 Results and Discussions…………………………….………………………..43

2.3.1 Enzymatic incorporation of alkylated purines opposite

an abasic site………………………………..………………………...43

2.3.2 Extension studies beyond an abasic site……………………………...50

2.3.3 Enzymatic incorporation of modified purine nucleotides opposite

templating DNA…..………………………………………………..…53

2.4 Conclusions…………………………………………………………………..61

CHAPTER 3 Evaluating the Base Stacking Contributions During Bypass of

Thymine Dimer Catalyzed by T4 DNA Polymerase…………………….……...63

3.1 Introduction………………………………………………………………….64

iv 3.2 Materials and Methods……………………………………………………….68

3.2.1 Protein purification, preparation of DNA substrates and synthesis

of non-natural nucleotides…………………………………………….68

3.2.2 Determination of the kinetic rate and dissociation constants for

dXTP incorporation opposite a thymine dimer……………………...69

3.2.3 Determination of rate limiting step using acid versus EDTA as

the quenching agent…………………………………………………..70

3.2.4 Idle-turnover measurements……………………………………………71

3.2.5 Exonuclease degradation of unmodified and damaged DNA………….71

3.2.6 Pyrophosphorolysis…………………………………………………….72

3.3 Results and Discussion………………………………………………………73

3.3.1 Non-natural nucleotides incorporation opposite a thymine dimer……75

3.3.2 Rate limiting step for replication of a thymine dimer…………………85

3.3.3 Exonuclease proofreading activity at a thymine dimer………………..90

3.3.4 Removal of natural and non-natural nucleotides

via pyrophosphorolysis………………………………………………95

3.4 Conclusions………………………………………………………………….98

CHAPTER 4 Spectroscopic Analysis of Translesion DNA Synthesis…..……………..104

4.1 Introduction………………………………………………………………...105

4.2 Materials and Methods……………………………………………………..108

4.2.1 Protein purification, preparation of DNA substrates and synthesis

of 5-NapITP ………………………………………………………108

4.2.2 Determination of kinetic rate constant for incorporation

v of 5-NapITP at an abasic site, thymine dimer and thymine

by rapid quench apparatus………………………………...……..109

4.2.3 Determination of kinetic rate constant for incorporation

of 5-NapITP at an abasic site, thymine dimer and thymine

using stopped flow apparatus..……………………………………109

4.2.4 Idle-turnover measurements…………………………………………..110

4.2.5 Pre-steady state kinetic analyses using Stopped Flow apparatus...…...111

4.2.6 Pre-steady state kinetic analyses using 32P assay..…………………...111

4.2.7 Measurement of exonuclease activity in the presence of next

correct nucleotide…………………….……………………………112

4.3 Results and discussion……………………………………………………...113

4.3.1 Enzymatic incorporation of 5-NapITP at an abasic site, thymine

dimer and thymine using gp43 exo-……………………………….115

4.3.2 Enzymatic incorporation of 5-NapITP at an abasic site, thymine

dimer and thymine using gp43 exo+ ………………………………119

4.3.3 Excision of 5-NapIMP at an abasic site, thymine dimer and thymine

by gp43 exo+ ………………………………………………………123

4.3.4 Pre-steady state analysis of 5-NapITP incorporation at

an abasic site by gp43 exo-……………………………….………..126

4.3.5 Experiments with next correct nucleotide…………………………...130

4.4 Conclusions…………………………………………………………………..134

CHAPTER 5 Conclusions and Future Directions………………………………………137

Appendix: Babho Devadoss‘s Publications…………………………………………….148

vi Bibliography……………………………………………………………………………195

vii LIST OF TABLES

CHAPTER 1

1.1 Kinetic parameters for dNTP incorporation opposite an abasic site………………...21 1.2 Kinetic parameters for the incorporation of 5-susbtituted indolyl nucleotides opposite an abasic site…………………………………………………..28 CHAPTER 2

2.1 DNA duplexes used to determine the kinetic parameters……………………………37

2.2 Summary of kinetic parameters for the incorporation of modified

nucleotides opposite an abasic site…………………………………………………49

2.3 Summary of kinetic rate constants for extension beyond an abasic site

catalyzed by gp43 exo- …………………………………………………………….51

2.4 Summary of kinetic parameters of the incorporation of modified

nucleotides opposite thymine and cytosine………………………………………...54

2.5 Summary of kinetic rate constants for the incorporation of modified

nucleotides opposite adenine and guanine…………………………………………58

CHAPTER 3

3.1 DNA duplexes used to determine the kinetic parameters……………………………68

3.2 Summary of kinetic rate and equilibrium constants measured for the insertion of

dATP and substituted indolyl-2’-deoxyriboside triphosphates opposite thymine

dimer and an abasic site……………………………………………………………77

3.3 Summary of kinetic rate and equilibrium constants measured for the

incorporation of dATP and 5-substituted indolyl-2’-deoxyriboside triphosphates

opposite a thymine dimer and thymine. ……………………………………………82

viii CHAPTER 4

4.1 DNA duplexes used to determine the kinetic parameters…………………………108

4.2 Summary of kinetic rate constants of 5-NapITP incorporation at an abasic site,

thymine dimer, and the thymine catalyzed by gp43 exo-……………….….……117

4.3 Summary of kinetic rate constants of 5-NapITP incorporation at an abasic site,

thymine dimer, and the thymine catalyzed by gp43 exo+…….……………..…..121

4.4 Summary of excision rate constants of 5-NapIMP at an abasic site, thymine dimer

and thymine catalyzed by gp43 exo+………………………………………….125

ix LIST OF FIGURES

CHAPTER 1

1.1 Chemical structures of four building blocks of DNA…………………………………3

1.2 Watson-Crick hydrogen base pairs……………………………………………………3

1.3 Bond angles and glycosylic bond distances between natural base pairs……………...4

1.4 Structures of base pairs existing in Watson-Crick, Hoogsteen, and Wobble

pairing arrangements………………………………………………………………..5

1.5 Schematic representation of DNA synthesis………………………………………….7

1.6 Proposed chemical mechanism for phosphoryl transfer catalyzed by DNA

polymerases…………………………………………………………………………8

1.7 Structural model of duplex DNA……………………………………………………..8

1.8 Kinetic mechanism of DNA polymerase……………………………………………..9

1.9 Crystallographic structure of klenow fragment……………………………………...12

1.10 Structures of non-natural base pairs………………………………………………..14

1.11 Structures of benzimidazole, 6-nitrobenzimidazole, 5-nitrobenzoimidazole

and 5-nitroindole………………………………………………………………….15

1.12 Structure of an abasic site and tetrahydrofuran……………………………………..18

1.13 Structure of pyrene triphosphate……………………………………………………25

1.14 Comparison of the structures of dATP with various 5-subsituted

Indolyl dexoxynucleotides………………………………………………………...26

1.15 Proposed model for DNA polymerization that invokes the

Contributions of π-electron interactions…………………………………………...30

CHAPTER 2

x 2.1 Chemical structures of modified nucleotides used in this study…………………….38

2.2 Kinetic mechanism of DNA polymerase activity…………………………………...41

2.3 Dependency of N6-methyl-dATP concentration on the

observed rate constant in primer elongation as measuring using

single-turnover conditions……………………………………………………….44

2.4 Experimental paradigm used to measure the insertion and extension

beyond an abasic site…………………………………………………………...50

2.5 Gel electrophoresis analysis showing the extension beyond an abasic

site catalyzed by gp43 exo-……………………………………………………….51

2.6 Graphical representation of modified nucleotides insertion of opposite

thymine or cytosine……………………………………………………………….55

2.7 Gel electrophoresis analysis of modified purines incorporation opposite

template containing adenine and guanine……………………………………….58

CHAPTER 3

3.1 Structures of thymine dimer and abasic site……………………………………..…65

3.2 Crystal structure of T7 DNA polymerase with DNA template containing

thymine dimer…………………………………………………………………..66

3.3 Chemical structures of non-natural nucleotides…………………………………….69

3.4 Screening of non-natural nucleotides incorporation opposite an abasic

site and thymine dimer………………………………………………………….73

3.5 Enzymatic incorporation of natural nucleotides opposite thymine dimer…………..74

3.6 Enzymatic incorporation of 5-NapITP opposite thymine dimer…………………….76

3.7 Enzymatic incorporation of 5-NITP opposite thymine dimer………………………79

xi 3.8 Proposed models for the enzymatic incorporation of non-natural

nucleotides……………………………………………………………………….83

3.9 Time courses for the incorporation of 5-PhITP opposite a

thymine dimer using EDTA or HCl as the quenching agent…………………….87

3.10 Time courses for the incorporation of 5-CHITP opposite a thymine

dimer, an abasic site and thymine using EDTA or HCl as the

quenching agent…………………………………………………………………88

3.11 Enzymatic hydrolysis of dAMP paired opposite a thymine dimer and

an abasic site by gp43 exo+……………………………………………………..91

3.12 Primer degradation of DNA containing a thymine dimer, an abasic site

or thymine by gp43 exo+……………………………………………………… 92

3.13 Idle turnover kinetics for 5-PhITP insertion opposite an abasic site

and a thymine dimer catalyzed by gp43 exo+…...……………………………..94

3.14 Pyrophosphorolysis activity on unmodified or damaged DNA templates

by gp43 exo- ………………………………………………………………….97

3.15 Proposed structure-activity relationships for the enzymatic incorporation

of various non-natural nucleotides opposite thymine dimer

versus an abasic site………………………………………………………….100

CHAPTER 4

4.1 Chemical structure of 5-NapITP…………………………………………………..105

4.2 Kinetic scheme representing the exonuclease proofreading pathway……………..106

4.3 Kinetic scheme illustrating the dynamics of nucleotide incorporation and

and excision by gp43 exo+ during translesion DNA synthesis………………...... 114

xii 4.4 Incorporation of 5-NapITP at an abasic site, thymine dimer and thymine

by gp43 exo- ………………………………………………………………….116

4.5 Time courses representing the incorporation of 5-NapITP opposite

an abasic site by gp43 exo- and gp43 exo+……………………………………120

4.6 Time courses representing the incorporation of 5-NapITP at an

abasic site, thymine dimer and thymine by gp43 exo+ ……………………….121

4.7 Time courses representing the incorporation and excision of 5-NapITP

opposite an abasic site by gp43 exo+…..……………………………………….124

4.8 Time courses representing the incorporation and excision of 5-NapITP

opposite an abasic site, thymine dimer and thymine using 32P assay…………125

4.9 Pre-steady state fluorescence quench time course representing the

incorporation of 5-NapITP opposite an abasic site……………………………129

4.10 Calibration curve to determine the enzyme concentration………………………..129

4.11 Pre-steady state time course representing the incorporation of 5-NapITP opposite

an abasic site measured using 32P assay……………………………………….130

4.12 Time courses of 5-NapITP incorporation at an abasic site in presence of next

correct nucleotide catalyzed by gp43 exo+……………………………………..131

4.13 Time courses of 5-NapITP incorporation at thymine in presence of next correct

nucleotide catalyzed by gp43 exo+……………………………………………..133

4.14 Proposed kinetic model for exonuclease proofreading mechanism of gp43 DNA

polymerase……………………………………………………………………..135

CHAPTER 5

5.1 Chemical structures of DNA adducts……………………………………………....142

xiii 5.2 Chemical structures of 8-oxoguanine, (6-4) photoproduct, and Dewar product…...143

5.3 Incorporation of natural nucleotides at template G by Pa-DinB……………………145

5.4 Incorporation of natural nucleotides at template containing 8-Oxoguanine and an

an abasic site…………………………………………………………………….145

5.5 Incorporation of non-natural nucleotides opposite an abasic site by Pa-DinB……..146

xiv ACKNOWLEDGMENTS

I am grateful to my major advisor, Dr. Irene Lee, for her guidance, continual

support and encouragement throughout my dissertation studies. I am thankful to have had

the privilege of being her student. I also thank my co-advisor Dr. Anthony J. Berdis for his scientific help, advice and his encouragement. He has been an excellent role model. I want to thank my colleagues, Dr. Xumei Zhang, Kevin Eng, Andrea Ramos, Joe

Schinmann for making the lab a dynamic environment to work and learn. I also want to thank my labmates, Dr. Diana Barko and Dr. Jessica Ward for taking time in teaching me spectroscopic techniques. Thanks to the Chemistry department and National Institute of

Health for their financial support. I remain grateful to all my friends for their help and support I have received during my stay in Cleveland. This thesis is dedicated to my parents, my wife and my brother for their unconditional love and support.

xv ABBREVIATIONS

32P radioactive phosphorus, with atomic weight 32

2-APTP 2-amino-2’-deoxyadenosine 5’-triphosphate

6-Cl-PTP 6-chloro-2’-deoxyadenosine 5’-triphosphate

6-Cl-2-APTP 6-chloro-2-amino-2’-deoxyadenosine 5’-triphosphate

7-deaza dATP 7-deaza-2’-deoxyadenosine 5’-triphosphate

7-deaza dGTP 7-deaza-2’-deoxyguanosine 5’-triphosphate

5-FITP 5-fluoro-indolyl-2’deoxyriboside triphosphate

5-AITP 5-amino-indolyl-2’deoxyriboside triphosphate

5-NITP 5-nitro-indolyl-2’deoxyriboside triphosphate

5-NapITP 5-napthyl-indolyl-2’deoxyriboside triphosphate

5-AnITP 5-anthracene-indolyl-2’deoxyriboside triphosphate

5-PhITP 5-phenyl-indolyl-2’-deoxyriboside triphosphate

5-CE-ITP 5-cyclohexene-indolyl-2’deoxyriboside triphosphate

5-CH-ITP 5-cyclohexyl-indolyl-2’deoxyriboside triphosphate DNA

deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphates dATP adenosine-2’- deoxyriboside triphosphate dGTP guanosine-2’- deoxyriboside triphosphate dTTP thymine-2’- deoxyriboside triphosphate dCTP cytosine -2’-deoxyriboside triphosphate

DTT dithiothreitol dPTP pyrene -2’-deoxyriboside triphosphate dsDNA double-stranded DNA

xvi

dH20 distilled water

dXTP non-natural deoxynucleoside triphosphate

dITP 2’-deoxyinosine 5’-triphosphate

EDTA ethylenediaminetetraacetic acid

gp43 exo- an exonuclease-deficient mutant of the bacteriophage T4 DNA

polymerase

gp43 exo+ wild type bacteriophage T4 DNA polymerase.

HCl hydrochloric acid kobs a rate constant

KD dissociation constant

kpol polymerization rate constant

kpol/KD catalytic efficiency

kexo a excision rate constant

Mg(OAC)2 magnesium acetate

N2-methyl dGTP N2-methyl-2’-deoxyguanosine 5’-triphosphate

N6-methyl dATP N6-methyl-2’-deoxyadenosine 5’-triphosphate

O6-methyl dGTP O6-methyl-2’-deoxyguanosine 5’-triphosphate

PNK polyribonucleoytidyl kinase

PMT photomultiplier tube on the stopped flow instrument

Pa-DinB Pseudomonas aeruginosa DNA polymerase

RNA ribonucleic acid

Sp space, as found in an abasic DNA site

Tris Tris(hydroxylmethyl)aminomethane

xvii TBE Tris-HCl/borate/EDTA

xviii Probing the Base Stacking Contributions during Translesion DNA synthesis

Abstract

by

BABHO DEVADOSS

Translesion DNA synthesis is the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. This process can cause mutagenesis resulting in disease development. During this process, DNA polymerases recognize

several biophysical features of the nucleotide during the incorporation opposite lesions to

promote mutagenic events. Using a series of non-natural nucleotides as molecular probes, we have evaluated the contributions of base stacking, shape complementarity, and solvation energies toward nucleotide incorporation and exonuclease proofreading activity of gp43 DNA polymerase at an abasic site and thymine dimer lesions. In chapter 2, we used modified purine nucleotides to provide evidence that the π-electron system and

hydrophobicity enhances the nucleotide incorporation at an abasic site, while the π-

electron system alone facilitates the extension beyond a mispair. In chapter 3, we

provided evidence that 5-NITP, a nucleotide that is preferentially incorporated opposite

an abasic site, yields a ~2,600-fold lower catalytic efficiency opposite a thymine dimer

compared to an abasic site and argues against the transient abasic site model proposed

earlier with T7 DNA polymerase and Dpo4. In contrast, 5-cyclohexene, 5-phenyl, and 5-

napthyl indole derivatives are incorporated 15-190-fold more efficiently than 5-NITP

opposite the thymine dimer. In general, the differences in catalytic efficiency reflect

xix perturbations in binding affinity in which nucleotides with larger π-electron surface area

bind more favorably than those lacking π-electron density during incorporation at the

thymine dimer. This is in contrast to an abasic site in which the binding affinity is

independent of π-electron surface area. Kinetic evidence suggests that the incorporation

of 5-PhITP and 5-CHITP opposite a thymine dimer is partially limited by the phosphoryl

transfer step while incorporation opposite an abasic site is limited by the conformational

change preceding phosphoryl transfer. In chapter 4, we used 5-NapITP as a fluorescent

probe to demonstrate the existence of pre-exonuclease complex during exonuclease

proofreading at either lesion. Our data suggest that the formation of the pre-exonuclease complex is driven by the base-stacking interactions with the lesions.

xx

CHAPTER 1

INTRODUCTION

1 1.1 The chemistry and biology of DNA

Dexoyribonucleic acid (DNA) is a biopolymer that carries genetic information in

humans and almost all other organisms. In general, DNA is considered to be blueprints

because it contains the essential instructions required for the building of cellular

components that include proteins and RNA molecules. At the molecular level, DNA is

composed of three basic components that include a nitrogen heterocyclic base, a pentose

sugar, and a phosphate group. The major heterocyclic nitrogen bases that act as main

building blocks are classified into two different categories: (i) monocyclic pyrimidines

including, thymine (T) and cytosine (C) and (ii) bicyclic purines that include adenine (A)

and guanine (G) (Figure 1.1).

At the core of nucleic acid structure is the concept of hydrogen bonding

interactions that are used to stabilize the nucleic acid and contribute to base pair

identification during DNA polymerization. In this case, the mutual recognition of A by T

and of G by C involves hydrogen bonding interactions between each partner. At the atomic level, the NH groups of the bases are good hydrogen bond donors, while the electron pairs on the oxygens of the base C=O groups and nitrogens on the ring are hydrogen bond acceptors. This can be best exemplified in Figure 1.2 showing the

hydrogen-bond acceptor and donor patterns for an A:T and G:C base pairs. This pattern is

commonly referred to as Watson-Crick base pairing.

In planar base pairs, the hydrogen bonding pattern yields a geometry that gives a

interglycosyl distance (C-1’ to C-1’) of 10.60 +/- 0.40 Å and an angle of 55o +/- 2o between the glycosylic bonds for both the A:T and G:C base pairs (Figure 1.3). The

2 isomorphous geometry indicates that all four base pair combinations A;T, T:A, G:C, C:G

can exist within the same regular framework of duplex DNA.

Figure 1.1 Chemical structures of four building blocks of DNA. dATP, dGTP, dCTP and dTTP are used as the building blocks during DNA synthesis by DNA polymerases.

dGTP dATP NH2 deoxy ribose guanosine triphosphate deoxy ribose adenosine triphosphate O N N N NH O O O N N O O O N N NH2 -O P O P O P O O -O P O P O P O O- O- O- O O- O- O- OH OH

dTTP deoxy ribose thymidine triphosphate dCTP O deoxy ribose cytidine triphosphate NH2 NH N O O O N O O O O O -O P O P O P O O N O- O- O- -O P O P O P O O - - - OH O O O OH

Figure 1.2 Watson-Crick hydrogen base pairs

NH O 2 O H2N N N N HN N NH N N N N N O dR dR N dR O dR NH2

A:T base pair G:C base pair

3 Figure 1.3 Bond angles and glycosylic bond distances between natural base pairs.

1.88Ao 1.78Ao N NH O CH3 N 2 O H2N 1.82Ao 1.86Ao N N HN N NH N N N N o N DNA O DNA 1.82A ooDNA NH O ~55 ~56 oo2 ~53 ~56 DNA o 10.6 +/- 0.3 A 10.2+/- 0.3 Ao

Major Groove Major Groove

N NH O CH3 N 2 O H2N

N N HN N NH N N N N N DNA O DNA DNA NH O 2 DNA Minor Groove Minor Groove

While Watson-Crick base pairing is the predominant pattern used to stabilize DNA, several other forms of base-pairing interactions have been identified. Two of the more commonly referenced forms include Hoogsteen and Wobble base pairs. Figure 1.4 compares the structural similarities and differences that exist between Watson-Crick,

Hoogsteen, and wobble base pairs. The Hoogsteen pair (Figure 1.4 B) is formed by one base rotating 180o relative to the other. This rotation makes the Hoogsteen pair not

isomorphous because the pair has an 80o angle between the glycosylic bond and a

separation of 8.6 Å of the anomeric carbons. In contrast, a "Wobble" base pair does not

involve rotation of a base but rather a sideways shift of one base relative to its position in

a normal Watson-Crick pair (Figure 1.4 C). In case of a wobble base pair formed between

4 G and T, the resulting loss of a hydrogen bond leads to a reduced stability, which may be

partially offset by improvements in base stacking interactions caused by the

displacement.

Figure 1.4 Structures of DNA base pairs existing in (A) Watson-Crick, (B) Hoogsteen, and (C) Wobble pairing arrangements.

O CH B N C 3 A N NH2 O CH3 NH2 N O CH3 N O HN N N N HN N HN N NH O N N DNA N N DNA O N DNA DNA O DNA DNA NH2

Although hydrogen bonding interactions are considered the most common physical feature associated with the conformation and dynamic structure of DNA, other biophysical parameters, such as desolvation, base stacking interactions and geometrical constraints, contribute extensively to the stability of nucleic acids (1). Desolvation refers to the energy required by the enzyme to remove the water molecules surrounding the heterocyclic bases while hydrophobicity refers to the tendency of a molecule to repel water. Although these terms are generally used interchangeably, they have different biophysical consequences toward stabilizing nucleobase interactions during DNA polymerization. As evident from the structures of duplex DNA, the interior of the helix is devoid of water and hence must be considered a hydrophobic environment. This hydrophobicity is necessary in order for direct hydrogen-bonding interactions to occur between the functional groups of the nucleobases. During the process of polymerization, it is clear that both the templating and incoming nucleobase must be desolvated to allow formation of the prerequisite hydrogen-bonding network within the hydrophobic core of

5 the DNA helix. Equally important are the π-stacking interactions among the aromatic bases. In the DNA helix, all nucleobases are stacked in an offset geometry that is effectively controlled by π−π electron interactions through their aromatic nature. It is important to emphasize that each of the aforementioned feature (hydrogen-bonding potential, π-electrons, and solvation energies) are interrelated such that one parameter influences the dynamic feature of the other. For example, it is difficult to alter a hydrogen bonding functional group without inadvertently influencing the solvation energy of the base or influencing its degree of aromaticity and/or base stacking capabilities.

The double helix of DNA resembles an intertwining spiral staircase in which the bases are stacked above one another. Each base is rotated ~36o around the helical axis relative to the next base pair. In this arrangement, about 10 base pairs make a complete turn of 360o. The twisting of the two strands to form the double helix creates a minor groove and a major groove. The minor groove is approximately 12 Å across while the major groove is roughly 22 Å in width. As indicated, both the major and minor grooves contain functional groups that can participate in hydrogen bonding interactions with proteins such as the DNA polymerase.

1.2 DNA synthesis

The synthesis of DNA is considered one of the most highly regulated and complex process in the biological system. Arguably, this process is crucial for correct maintenance of the genomic information for cellular survival. In many organisms, this process is highly regulated through the concerted action of dozens of proteins that include the DNA polymerases. During the replication process, DNA polymerases add

6 mononucleotides into a growing primer using DNA template as the guiding template

information for the correct nucleotide incorporation (Figure 1.5).

Figure 1.5 Schematic representation of DNA synthesis.

OH DNA polymerase OH ACGT + dNTP ACGTN + PPi TGCAYCTA TGCAYCTA

The chemistry of this process is a simple transesterification reaction in which the α-

phosphate of the incoming 5'-deoxynucleoside triphosphate (dNTP) undergoes

nucleophilic attack by the 3'-OH of the primer strand of the nucleic acid (2). The reaction is catalyzed with the participation of active site carboxylate residues that coordinate two metal ions. It is proposed that one metal ion aids in the abstraction of the proton from the

3'-OH (3),while the other participates as a Lewis acid to coordinate the β−γ phosphate groups of the incoming dNTP to form the complex DNA biopolymer (Figure 1.6).

7 Figure 1.6 Proposed chemical mechanism for phosphoryl transfer catalyzed by DNA polymerases

H C H2C 2 H2C CH CH2 CH 2 C 2 C -O C C - C - -O O - -O O O C - -O O O O P O -O O O O O O O 5' O O 2+ O 2+ P 2+ 5' O Mg 5' O Mg O O P O O Mg O P O O P O O O O P O O O- O P - P 2+ P O O- O O O Mg O OH O P -O OH -O -O O- O- O O O O O O O O O A A A C C C A A T T T G G T G T T O O O O O O O O O - O - - - 3' O - O - O - O O O - 3' O- 3' O O P O O P O P O O P O O O P O P O O P O O O P O O P O O O O O O O O O O O O O

The well-coordinated process results in formation of error-free long DNA biopolymer that consists of two complementary DNA strands to form a double helix

(Figure 1.7). At the core, the integrity of the DNA helical structure is maintained through classical Watson-Crick hydrogen bonding and steric interactions (4).

Figure 1.7 Structural model of duplex DNA

8 During DNA synthesis, several kinetic steps are involved to ensure the faithful generation of multiple copies of DNA by DNA polymerase. A detailed proposed kinetic scheme is illustrated in Figure 1.8. In this process, the first point for generating catalytic efficiency and fidelity of nucleotide incorporation occurs during the binding of dNTP to the polymerase:DNA complex (Step 2). After dNTP binding, a conformational change

(Step 3) in the enzyme is proposed to align the incoming dNTP into a precise geometrical conformation for phosphoryl transfer (Step 4). Results from several independent experiments, including pulse-chase (5) and fluorescence quenching techniques (6) indicate that the rate constant of ~100 sec-1 measured with the T4 DNA polymerase corresponds to a conformational change step preceding phosphoryl transfer. This kinetic step is the major contributor to polymerization fidelity as it constitutes a key kinetic point for error discrimination.

Figure 1.8 Kinetic mechanism of DNA polymerase

dNTP 2 Conformational DNA Binding E:DNA E:DNA :dNTP Change n n 3 1 dNTP Binding E':DNAn:dNTP Processive 8 DNA Synthesis Phosphoryl E + DNAn Transfer 4

7 E':DNAn+1:PPi Enzyme PPi 5 Dissociation 6 Conformational E:DNAn+1 E:DNAn+1:PPi Change Pyrophosphate Release

The involvement of the conformational change is consistent with the proposed "induced- fit" mechanism that imposes discrimination against the misinsertion of a dNMP since

9 misaligned intermediates may disrupt or alter the geometry of the polymerase’s active site to prevent the chemical reaction (7-9). It is also possible that error discrimination can

occur during the phosphoryl transfer step (Step 4) such that it becomes completely rate-

limiting (10). Indeed, it is proposed that phosphoryl transfer is completely or partially rate-limiting for nucleotide incorporation with pol β (11) and the RNA-dependent RNA

polymerase from poliovirus (12).

The molecular events underlying this kinetic mechanism have historically been

interpreted with the formation of hydrogen bonds between base pairs (13). In this model,

the incoming dNTP is proposed to pair directly opposite its complementary template

partner so that the enzyme catalyzes the transesterification reaction only if the alignment between the two is correct. As such misincorporation events are predicted to occur rarely because of the inability of the functional groups to line up properly that either perturbs

ground state binding (Step 2) and/or reduces the rate of the conformational change (Step

3). At face value, the kinetic constants are consistent with this model. For example,

misincorporation of dATP opposite C catalyzed by the bacteriophage T4 DNA

polymerase is disfavored ~330,000-fold compared to correct incorporation of dATP

opposite T (14). The binding affinity for dATP opposite C is 1,100 μM and is ~110-fold higher than the binding affinity of 10 μM measured for dATP incorporation opposite T

(15). In addition, the rate constant for the conformational change preceding the chemical

-1 reaction step is ~0.03 sec and represents a 3,300-fold reduction compared to the kpol

value of 100 sec-1 measured for the correct incorporation of dATP opposite T (15). Thus,

the catalytic efficiency for creating a mismatch is 27 M-1sec-1 while that for correct DNA

synthesis is 107 M-1sec-1. The differences in catalytic efficiencies of 2.7*10-6 correspond

10 to a predicted error frequency of one mistake in every 1,000,000 incorporation events.

This calculated error frequency is close to that measured in vivo with the exonuclease

deficient T4 DNA polymerase (16).

An additional level of error discrimination results from the proofreading of any

formed mismatches by the 3’Æ 5’ exonuclease activity of the polymerase. This activity

removes misincorporated nucleotides to regenerate a correctly base-paired

primer/template and can reduce the error frequency by an additional 102-103-fold (17).

1.3 Structural features of DNA polymerases

Kinetic characterizations of DNA polymerases have provided the basic framework for understanding the dynamics of correct and incorrect DNA synthesis.

However, it was not until 1985 that the first detailed structure of a Klenow fragment

DNA polymerase was reported by Tom Steitz’s group (18). Crystallographic snapshots of several DNA polymerases have later been reported. These include the structures of pol β,

HIV-RT, bacteriophage T7 and bacteriophage RB69. The comparative analysis with the structural features revealed that all DNA polymerases share a shape of a half-opened right hand, with three distinct subdomains designated as the palm, finger and thumb domains. (Figure 1.9) Finger domain conformation is again subdivided into open or closed conformation based on the position. In the open conformation, the template strand is bound primarily by the finger and palm domain, positioning the template:primer junction at the polymerization site, found in a cleft between these subdomains.

11 Figure 1.9 Crystallographic structure of Klenow fragment. The cartoon structure is drawn using protein explorer using the Pdb 1SKS from the protein databank.

Finger Thumb Domain Domain

Palm Domain

Based upon available sequence alignment data and crystallographic analyses,

DNA polymerases can be divided into at least five distinct families denoted as A, B, X,

Y, and reverse transcriptase (19). Among the different families, the most extensively studied polymerase family is the A family which includes the Klenow fragments of DNA polymerase I from Escherichia coli (20) and Bacillus subtillus (21), Thermus aquaticus

DNA polymerase (22), and the bacteriophage T7 DNA polymerase (23). These structures indicate that all DNA polymerases share a common overall architecture resembling a

"right hand" containing fingers, palm, and thumb subdomains. The palm domain is responsible for catalysis of the phosphoryl transfer reaction and, as such, is the most closely conserved structural feature among all polymerases. The palm domain contains at least two carboxylates that function to coordinate two catalytically essential metal ions

12 that participate in phosphoryl transfer. The fingers domain interacts with the incoming

dNTP as well as the templating base, and thus plays an important role in maintaining

fidelity. The thumb domain plays dual roles by positioning duplex DNA for the incoming

dNTP as well as in the processivity and translocation of the polymerase. Although the

functions of the fingers and thumb subdomains are relatively conserved among DNA

polymerases, they are widely divergent of structural characteristics. These structural

differences are proposed to account for nuances in the dynamics of nucleotide

incorporation and intrinsic fidelity of various polymerases (24).

The structures of several DNA polymerases complexed with nucleic acid in the

absence and presence of dNTP reveal features consistent with the mechanism described

in Figure 1.8 determined through kinetic studies. In these models, the dNTP binds to the

polymerase:DNA complex in a template-independent manner through interactions with

the fingers domain. This initial binding event is followed by a conformational change that

corresponds to rotations of the fingers domain that then induces "tighter" binding with the templating nucleobase. One hypothesis is that this rotation provides the driving force to align the incoming dNTP with its complementary partner and/or to orient the 3’-hydroxyl of the primer for attack on the bound dNTP.

1.4 Models accounting for nucleotide selectivity during DNA polymerization

1.4.1 Active site tightness as a model for DNA polymerization

Historically, it is considered that DNA polymerase activity depends heavily

on Watson-Crick hydrogen bonding between the base pairs. Once the nucleotide is

bound to the enzyme-active site, the remaining kinetic steps then would allow for the

primer elongation. However, evidences from thermal denaturation studies of nucleic acid

13 duplexes with properly and mispaired nucleobases illustrated that the energy differences

were too small to account for the observed low error rates. For example, as for the

hydrogen bonding model, the nucleotide binding step is the one where the discrimination

must take place. Because misincorporation occurs in only 1 in 106 polymerization events,

the ratio of the KM values for improper to precise polymerization should also be about

6 10 . More specifically, the KM for the pol α incorporation of A opposite T is 3.7 +/- 0.7

μM. For the misincorporation of G opposite T, a KM of 4,200 +/- 900 μM was determined. The ratio of KM values (4200/3.7 = 1135) is almost two orders of magnitude

too low to account for the overall observed fidelity. This study indicates that if

nucleotide discrimination only occurred during nucleotide binding, the error rate would

be about 1 in 104.

To address this issue, Eric Kool’s laboratory designed a base pair consisting of

4-methylbenzimidazole and 2,4-difluorotoluene that are completely devoid of hydrogen

bonding groups. As illustrated in Figure 1.10, the base pair comprised of 4-

methylbenzimidazole opposite difluorotoluene is proposed to be geometrically identical

to an adenine:thymine base pair(25).

Figure 1.10 Structures of non-natural base pairs

Adenine 4-Methylbenzimidazole H H F N N F CH3 N CH3 CH3

N N N dR N dR F F dR dR 2,4 Difluorotoluene 2,4 Difluorotoluene

14 Kinetic studies reveal that the overall catalytic efficiency for the incorporation of 2,4-

difluorotolune opposite 4-methylbenzimidazone is 200-fold lower than that for the

incorporation of dTTP opposite A (25). Collectively, this evidence indicates that biophysical factors other than hydrogen bonding must be considered when evaluating the mechanisms that contribute to DNA polymerase fidelity.

1.4.2 Negative selection as model for nucleotide incorporation.

To further delineate the underlying mechanism, Kutcha’s laboratory measured the incorporation of several purine analogs such as benzimidazole, 5- nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole (Figure 1.11) (26).

Figure 1.11 Structures of benzimidazole, 6-nitrobenzimidazole, 5-nitrobenzoimidazole and 5-nitroindole

Benzimidazole 5-Nitrobenzimidazole 6-Nitrobenzimidazole 5-Nitroindole NO N N NO2 N 2

N N N NO2 N R R R R

The kinetics of nucleotide incorporation was measured using three different polymerases that include the eukaryotic pol α, the E.coli Klenow fragment, and the

Moloney-murine leukemia virus (MMLV) reverse transcriptase. MMLV reverse transcriptase was unable to incorporate any of the non-natural nucleotides opposite templating nucleobases or an abasic site, a non-templating DNA lesion (26). The lack of incorporation apparently reflects the inability of the reverse transcriptase to catalyze phosphoryl transfer because the non-natural nucleotides can prevent the binding of

15 natural dNTPs to the polymerase to inhibit replication (26). These results suggest that the

MMLV reverse transcriptase does not require hydrogen-bonding groups for nucleotide binding but instead utilizes these interactions to facilitate phosphoryl transfer. The most striking result of their study is the lack of shape complementarity observed during the incorporation of the various nitrobenzimidazole opposite their predicted partners.

According to the shape complementarity model, benzimidazole analogs should be preferentially incorporated opposite C and T. However, in nearly all cases examined,

Klenow fragment preferentially inserted these non-natural purine analogs opposite natural purines rather than pyrimidines. Similarly, pol α also incorporates some nitrobenzimidaoles opposite A and G with nearly identical catalytic efficiencies compared to their incorporation opposite C and T. Based on these data an alternative model for DNA polymerization was proposed that invokes "negative selection" of inappropriate nucleotides as the predominant force used to maintain replication fidelity

(26). The negative selection model postulates that during the binding of dNTPs, the polymerase allows the nucleobase of the dNTP and the template base to adopt the lowest free energy conformation during their interaction. If the interactions are favorable, the polymerase then allows the conformational change to occur to facilitate phosphoryl transfer. However, incorrect pairing prevents the conformational change, and thus does not allow catalysis to occur. Through this mechanism, the polymerase can sample a wide variety of nucleotides. However, only the dNTP that adopts the lowest free energy conformation will be accepted due to the formation of favorable interactions. This model differs from most positive selection models that postulate that the polymerase is allowed to sample only a limited number of possibly correct partners. Though these data indicate

16 that the hydrogen bonding interactions are not needed to form a base pair, they also indicate that the “steric fit” model is not universal. Thus, the ability to form a “correctly” shaped base pair is not an absolute requirement for nucleotide incorporation.

Models involving desolvation and hydrophobicity as primary determinants for the fidelity were put forward by Goodman and Petruska (27). However, it has become

nearly impossible to unambiguously evaluate the role of desolvation as a mechanism of

polymerase fidelity. For example, altering the functional groups of a natural nucleotide

affects the overall hydrogen bonding potential and tautomeric form of the nucleotide.

Thus, changing one biophysical feature inadvertently influences another.

In conclusion, a model to account for all the observations surrounding DNA

polymerase fidelity is still evolving. There are several forces that appear to be involved.

An alternate approach is the use of damaged DNA, was employed by several laboratories

including our lab to further evaluate the role of hydrogen bonding interactions, steric

constraints and base-stacking contributions.

1.5 Translesion DNA synthesis

Translesion DNA synthesis is the ability of a DNA polymerase to incorporate a

nucleotide opposite damaged DNA and extend beyond the damaged site to propagate a

potential genetic mistake. Perhaps the most prevalent and pro-mutagenic class of DNA

lesion is an abasic site that is considered to be the prototypical non-coding DNA lesion

(Figure 1.12). Abasic sites are formed by the hydrolysis of the bond between the C1' of

ribose and the N9 of a purine or the N1 of a pyrimidine (28). Studies from Lindahl

laboratory have shown that at least 10,000 abasic sites are formed on a daily basis in a

given cell making it one of the vastly studied lesions (28).

17 Although abasic sites arise spontaneously under normal physiological

conditions, their formation is enhanced by exposure to ionizing radiation and certain

chemicals (29) as well as through the action of DNA glycosylases, which act to excise

damaged nucleobases (30). Most abasic sites are repaired by the base excision repair

pathway (31). However, a small fraction of lesions can persist and be replicated to yield a

high probability of mutagenesis. Because no coding information is present at an abasic

site, there exists a potential to incorporate all four dNTPs with equal efficiency. This situation could result in a 75% chance of incorporating the wrong nucleotide to cause a genetic mutation. Alternatively, the lack of coding information could provide a strong roadblock and prohibit movement of the DNA polymerase to stop DNA replication completely.

Figure 1.12 Structure of an abasic site and tetrahydrofuran

O

N NH

N Tetrahydrofuran O N NH2 Abasic site O

O O O O O OH OH H O Depurination O - O O P O O O - O P O- - O P O O O P O O O O

1.6 Incorporation of natural nucleotides opposite an abasic site

Despite the lack of coding information at an abasic site, DNA polymerases such as eukaryotic pol α (32) and pol δ (33), the E. coli DNA polymerase I (34), the

18 bacteriophage T4 DNA polymerase (35), and HIV reverse transcriptase (36)

preferentially insert dAMP (and dGMP to a lesser extent) opposite an abasic site. This

kinetic phenomenon is commonly referred as the "A-rule" of translesion DNA synthesis

(37). The mechanism for the "A-rule" is enigmatic since the ability of a polymerase to

incorporate preferentially one specific nucleotide opposite this non-templating lesion

cannot be reconciled by models invoking hydrogen-bonding and/or shape

complementarity interactions. In an attempt to define this mechanism, the Goodman

laboratory investigated the incorporation of natural dNTPs using pol α isolated from

Drosophila (32). Their kinetic analyses revealed that amongst the four natural

nucleotides, dATP is incorporated the fastest, and is 5-fold faster than the Vmax using

dGTP (32). In addition, the KM for dATP is 1.4 mM while that for dGTP is 3.4 mM, and

these values are significantly lower than those measured for the pyrimidines, dCTP and

dTTP (32). Of course, these values are significantly different than those measured during normal DNA synthesis. Specifically, the KM value for a correct dNTP opposite its

complementary partner is ~10-fold lower than that measured for incorporation opposite

an abasic site. Likewise, the Vmax for incorporation opposite the abasic site is

significantly lower than that for normal DNA replication.

An important feature of these studies was the quantitative evaluation of nearest neighbor effects on the dynamics of nucleotide selection (32). With pol α, the Vmax for dATP incorporation varies as the penultimate base is changed. In fact, the highest Vmax

value is measured with T as the penultimate base while the lowest is observed with C

(32). At face value, these results suggest that the preferential incorporation of dATP

arises through hydrogen-bonding interaction with the nearby templating base. However,

19 this is not accurate because the Vmax for dGTP incorporation opposite an abasic site is also highest when T is the penultimate base rather than C.

Using the E. coli DNA polymerase I, Livneh's group reported that dATP is also preferentially incorporated opposite an abasic site (34). In this case, the KM for dATP is

32 μM (34) and is only 3-fold worse than the KD of 10 μM measured for the

-1 incorporation of dATP opposite T (38). The kcat value of 0.059 s for dATP opposite an

-1 abasic site is likewise reduced 850-fold compared to the kpol of 50 sec measured for

dATP opposite T (38). Similar to pol α, the E. coli polymerase preferentially utilizes

purines as opposed to pyrimidines. In general, the KM values for purines are ~5-fold

lower than those measured for dCTP or dTTP while the kcat for dATP is faster than that

for the other three natural nucleotides.

Low fidelity polymerases have also been tested for their ability to perform

translesion DNA synthesis. Goodman and coworkers demonstrated that HIV reverse

transcriptase is similar to high fidelity polymerase because it also preferentially

incorporates dATP opposite an abasic site (39). With this enzyme, the Vmax/KM value for dATP incorporation opposite the abasic site is 5-fold higher than for the insertion of dGTP and 15- and 20-fold higher than dTTP and dCTP, respectively (39). However, the preferential incorporation of dATP opposite the abasic site is not universal because certain error-prone DNA polymerases prefer to incorporate other natural nucleotides. For example, pol iota preferentially incorporates dGTP opposite this lesion (40).

20 Table 1.1 Kinetic parameters for natural nucleotides incorporation opposite an abasic site by T4 DNA polymerase.

-1 dNTP KD (μM) kpol (sec ) dATP 35 +/- 5 0.15 +/- 0.01 dCTP 250 +/- 50 0.010 +/- 0.001 dGTP 130 +/- 20 0.02 +/- 0.01 dTTP 1200 +/- 200 0.06 +/- 0.01

In fact, the polymerase appears to discriminate against dATP incorporation as dGTP and

dTTP are incorporated with higher efficiencies than dATP (40). Another polymerase that defies the "A-rule" is the Rev 1 polymerase from yeast that preferentially incorporates dCTP opposite the abasic site (41). These examples appear to reflect extreme behavior as both polymerases show a distinct preference for utilizing one specific dNTP. Rev 1, an error prone polymerase also incorporates dCTP opposite other DNA lesions that are not

predicted to be the complementary partner of cytidine (41).

Previous works from Berdis lab using the bacteriophage T4 DNA polymerase arguably provide the most comprehensive evaluation of the underlying mechanism of

nucleotide selection during translesion DNA synthesis catalyzed by a high fidelity

polymerase. Using single turnover reaction conditions (enzyme in molar excess versus

DNA substrate), Berdis lab were able to define the kinetic dissociation constant, KD, as

well the maximal polymerization rate, kpol, for all four natural dNTPs opposite an abasic

site (35). As summarized in Table 1.1, dATP is preferentially incorporated compared to

other dNTPs. This enhancement arises because dATP binds with higher affinity. In this

case, the KD value of 35 μM for dATP is 4- to 34-fold lower than the other natural

21 -1 nucleotides. In addition, the kpol value for dATP is 0.15 sec and is between 3- to 10-fold

faster than that measured for the other dNTPs.

Using these values, the catalytic efficiency for the incorporation of dATP located

across from an abasic site is calculated to be 4.3x103 M-1s-1. This value is significantly

lower than that of 107 M-1s-1 measured for the correct incorporation of dATP opposite T

(35). Surprisingly, the decrease in catalytic efficiency for translesion synthesis arises

primarily from a reduction in the rate of the conformational change (~700-fold) and only a minimal change in ground state binding (~ 3-fold). Another significant difference in the

dynamics of incorporation reflects alterations in the time course in product formation

measured under pseudo-first order reaction conditions (DNA and nucleotide in molar

excess versus polymerase). Using unmodified DNA, the time course for dATP

incorporation opposite T is biphasic and indicates that a step after phosphoryl transfer is

rate-limiting for polymerase turnover (35). In contrast, the time course for dATP

incorporation opposite the abasic site is linear, and indicates that phosphoryl transfer or a

step before the phosphoryl transfer is the rate-limiting step (35). Furthermore, the lack of

a significant elemental effect when α-S-dATP was substituted for α-O-dATP suggests that the phosphoryl transfer step is not rate-limiting for incorporation opposite the DNA lesion (35). Thus, the data from several independent experiments suggest that the conformational change step preceding phosphoryl transfer limits the overall rate of nucleotide incorporation opposite an abasic site.

These studies with the bacteriophage T4 DNA polymerase revealed that the larger purines are inserted with an overall higher catalytic efficiency (kpol/KD) compared

to the smaller pyrimidines (35). The higher catalytic efficiency for the larger nucleotides

22 is consistent with the shape complementarity model proposed by Kool's group (42).

However, there exist other alternatives, including the effects of nucleobase desolvation

and base-stacking capabilities rather than the absolute size and/or shape of the molecule.

Because adenine has relatively low solvation energy, the preferential insertion of dATP

opposite an abasic site could also reflect the influence of desolvation.

An alternative model is the base-stacking model that is based upon the correlation

between the measured kpol/KD values of dNTPs and their relative conformation when

placed opposite an abasic site (43). NMR studies indicate that dAMP exists in a

thermodynamically favored intrahelical conformation when placed opposite the lesion

(43). This favored conformation correlates well with the relatively high kpol/KD value for

dAMP insertion. On the other hand, dCMP and dTMP are found to exist in an

unfavorable extrahelical position (128) that coincides with their overall poor kpol/KD values. These observations led Berdis lab to propose that the efficiency of nucleotide insertion directly reflects the “stacking” capabilities of the nucleobase (35).

The dynamics of this model were tested by correlating the ability of the T4 DNA polymerase to extend beyond an abasic site with the proposed helical position of the nucleobase paired opposite the lesion. It was proposed that an intrahelical base such as adenine should be extended much more efficiently than an extrahelical base such as cytosine or thymine (35). As predicted, the rate of extension from adenine paired opposite an abasic site is 20-fold faster than extension beyond a G:abasic site mispair and at least

100-fold faster than extension beyond a C:abasic mispair. Collectively, these data argue that the efficiency of extension beyond a dNMP:abasic site is modulated by whether the terminal base pair exists in an intra- or extrahelical conformation. Structural studies of the

23 bacteriophage T4 homologue poised at an abasic site provide further evidence for this model as adenine is observed in a thermodynamically favored conformation inside the helical structure of duplex DNA (44).

1.7 The use of non natural nucleotides to probe shape complementarity during translesion

DNA synthesis

Several laboratories used non-natural nucleotides as probes to investigate the dynamics of translesion DNA synthesis. One of the earliest endeavors in this field is work by Kool’s laboratory that investigated the role of shape complementarity during replication opposite an abasic site (42). The shape complementarity model predicts that nucleotides possessing the correct shape and size of a "true" base pair should be incorporated with higher catalytic efficiencies than those lacking these requirements.

Indeed, the validity of this model has been strengthened by the demonstration that pyrene triphosphate (Figure 1.13) is inserted opposite an abasic site at least 100-fold more efficiently than any of the four natural dNTPs (42). Using the E. coli Klenow fragment,

Morales and Kool demonstrated that the KM of 3 μM for pyrene triphosphate incorporation opposite the lesion is 27-fold better than the Km of 85 μM measured for

-1 dATP (42). Likewise, the kcat of 9 sec for pyrene triphosphate is 10-fold faster than that measured for the incorporation of dATP (42). The facile insertion of pyrene triphosphate opposite the abasic site suggests that the "void" could be easily filled by the bulky aromatic nucleobase. Indeed, modeling studies revealed that the overall shape and size of the pyrene:abasic site mispair is nearly identical to that of the natural adenine:thymine pair

(42).

24 Figure 1.13 Structure of pyrene triphosphate

O O O

-O P PO O P O O O- O- O- OH

The lack of hydrogen-bonding potential of either the abasic site or the incoming pyrene are consistent with the shape complementarity model. However, other factors such as base-stacking and desolvation could also play a significant role in the incorporation of pyrene triphosphate opposite this DNA lesion. In addition, pyrene triphosphate was also incorporated opposite a templating nucleobase only 100-fold less efficiently compared to incorporation opposite an abasic site (42). The lower efficiency is caused by reductions in

Vmax which are ~1% when measured for incorporation opposite the lesion. Surprisingly, the KM for pyrene triphosphate is essentially identical (>10 μM) regardless of templating base (42). Thus, while the alterations in Vmax values can be explained by simple steric exclusion, the ability of the polymerase to bind the large, bulky nucleotide rather tightly regardless of shape complementarity argues that other physicochemical parameters may influence its incorporation.

25 1.8 The role of π-electron surface area during translesion DNA synthesis.

Our lab took the initiative to evaluate the extent of contributions of shape

complementarity, base-stacking, and solvation toward nucleotide incorporation by

measuring the insertion of a series of modified nucleotides opposite an abasic site.

Figure 1.14. Comparison of the structures of dATP with various 5-substituted indolyl deoxynucleotides.

NH2 O F NH +N N N 2 O-

N N N N N N dR dR dR dR dR dATP IndTP 5-FITP 5-AITP 5-NITP

N N N dR dR dR 5-CHITP 5-CEITP 5-PhITP

O O O dR= -O P O P O P O O O- O- O- OH

Our initial studies identified a unique non-natural nucleotide, 5-nitro-indolyl-2’-

deoxyriboside triphosphate (5-NITP) (Figure 1.14), that was inserted opposite an abasic

site with approximately 1,000-fold greater efficiency compared to that for dATP insertion

(45). Kinetic characterization reveals that 5-NITP is incorporated opposite an abasic site

-1 with a fast kpol value of 126 sec and a low KD value of 18 μM. Remarkably, these values are nearly identical to those for the enzymatic formation of a natural Watson-Crick base pair (5) and reiterate the finding that hydrogen bonding potential is not essential for

26 nucleotide insertion (46). Although 5-NITP is also inserted opposite natural nucleobases with varying degrees (45), the catalytic efficiency is 30-fold greater for translesion DNA synthesis. The high catalytic efficiency results from an enhancement in the conformational change step preceding phsophoryl transfer (high kpol value) rather than an enhancement in binding affinity (identical KD values).

The importance of the nitro moiety was first evaluated by simply replacing this functional group with -H ((47). This replacement reduces the catalytic efficiency for nucleoside insertion by ~2,300-fold (47). Although binding affinity is perturbed 8-fold, the major effect of this substitution is the 450-fold reduction in the rate of the conformational change step that corresponds to a change in relative free energy (ΔΔG) of

3.62 kcal/mol. This energetic difference is proposed to arise through base-stacking interactions mediated between the overlapping π-electron densities of the conjugated indole nucleoside with the polymerase and DNA. This model is further strengthened by the crystal structures of RB69 crystallized with 5-NITP opposite an abasic site by

Doublie. However, other forces such as desolvation/hydrophobicity as well as size and shape complementarity cannot be unambiguously refuted based solely upon these data.

To address the influence of these molecular forces, analogs corresponding to 5- fluoro-, 5-amino-, 5-cyclohexyl, 5-cyclohexene, and 5-phenyl-indole-2’deoxyriboside triphosphates (Figure 1.14) were synthesized and characterized for incorporation opposite an abasic site (48). KD and kpol values for their incorporation are summarized in Table

1.2. The 5-fluoro- and 5-amino- analogs are similar to the indole derivative as all three are poorly inserted opposite the abasic site (49). The high KD values of 150-200 μM and

27 -1 the low kpol values of ~0.2 sec for these analogs arguably reflect the loss of energetic stabilization caused by the lack of π-electrons present at each substituent group.

The most interesting trend is observed for the incorporation of 5-cyclohexyl, 5-

cyclohexeneyl, and 5-phenyl-indole-2’deoxyriboside triphosphates. The bacteriophage

T4 DNA polymerase incorporates the 5-phenyl derivative opposite an abasic site with an

-1 extremely high catalytic efficiency. The kpol of 53 sec and KD of 14 μM are nearly identical to those measured for the 5-nitro analog (48) and suggests that analogs containing conjugated functional groups bind to the polymerase with high affinity and are inserted faster opposite the abasic site.

To test this mechanism, the isosteric analogs, 5-cyclohexyl and 5-cyclohexeneyl, were tested for incorporation opposite an abasic site (48). The catalytic efficiency for the

5-cyclohexeneyl-indole analog is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects a substantial increase in the kpol value (compare

25 versus 0.7 sec-1, respectively) rather than an influence on ground-state binding. The

faster kpol value for the 5-cyclohexeneyl-indole derivative indicates that π-electron density enhances the rate of the enzymatic conformational change step required for insertion opposite the abasic site.

Table 1.2 Kinetic parameters for the incorporation of 5-substituted indolyl nucleotides opposite an abasic site by T4 DNA polymerase.

-1 dXTP KD (μM) kpol (sec ) 5-NITP 18 +/- 3 126 +/- 7 IndTP 145 +/- 10 0.28 +/- 0.07 5-FITP 152 +/- 41 0.30 +/- 0.03 5-AITP 255 +/- 40 0.17 +/- 0.01 5-PhITP 14 +/- 3 53 +/- 4 5-CEITP 5.1 +/- 1.7 25 +/- 2 5-CHITP 44 +/- 14 0.70 +/- 0.13

28 However, the near identity in KD values suggests that biophysical parameters

independent of π−electron density such as desolvation and/or size also influence dNTP

binding

1.9 An alternate model for DNA polymerization

The results of the kinetic analyses were interpreted with respect to the physical

properties of each nucleotide analog in conjunction with available structural information

of various DNA polymerases. An essential caveat of this model is that the templating

nucleobase is oriented in a conformation that prevents direct hydrogen-bonding contacts between the templating base and the incoming nucleotide during the initial binding event

(49). Since the orientation of the templating nucleobase is in an extrahelical position, a transient "void" is created in the DNA that provides a functional mimic of an abasic site

(Figure 1.15, step A). It is easy to envision that large, bulky nucleotides such as 5-phenyl

and 5-cyclohexyl-indoles can easily fill the "void" produced by the transiently-formed

abasic site intermediate. Because both molecules are similar in shape and size, their KD values are essentially identical and independent of templating nucleobase. Unlike binding affinity, however, the kpol value is highly dependent upon the presence of a templating

nucleobase.

29 Figure 1.15. Proposed model for DNA polymerization that invokes the contributions of π-electron interactions.

-O O- O P R O - P O O O O N O O -O OP OP P O O - P O O- O- O- O O OH OH O Kd N

R Step A

kpol Step B

O- -O - P O O P O- O O P O O k O OH OH chem O O N N

Step C R R

We argue that the kpol step (Figure 1.15, step B) represents the conformational change step required to place the templating base in an intrahelical conformation that then allows for proper alignment of the primer-template required for the phosphoryl transfer step

(kchem) (Figure 1.15, step C). At an abasic site, the rate of this conformational change is significantly increased because the lack of a templating nucleobase circumvents the need for this re-positioning. Indeed, it is notable that the non-natural nucleotides are poorly incorporated opposite templating nucleobase (49). This result argues that the rate of this conformational change step is slowed when a templating base is present. This most likely is caused by the shear bulk of the non-natural nucleotide hindering the facile re- positioning of the templating nucleobase from an extra- into an intra-helical conformation.

30 It should be emphasized that this model differs from the shape complementarity

model for several reasons. First, the shape complementarity predicts that large non- natural nucleotides such as 5-phenyl and 5-cyclohexyl-indoles should both be effectively inserted opposite the abasic site because both provide the proper size and shape to adequately fill the void. Despite having similarities in shape and size, however, the kpol value for the cyclohexyl analog is ~100-fold slower than that for the cyclohexeneyl analog. The most discernible difference between these two analogs is with respect to π- electron density. Thus, while dNTP binding opposite the DNA lesion appears to be influenced by shape/size constraints in addition to the hydrophobicity of the incoming nucleotide, the rate of the conformational change is directly linked with the presence of

π-electron density at the 5 position of the modified indole. At the atomic level, Berdis lab proposed that this stabilization is mediated through interactions with aromatic amino acids present in the active site of the DNA polymerase (45). The enzyme-mediated conformational change transfers the aromatic nucleobase into the aromatic environment of the duplex DNA and provides entropic stabilization.

1.10 Statement of purpose

We now know that T4 polymerases use non-natural nucleotide that is completely devoid of any functional groups during the incorporation event opposite an abasic site.

However it is clear that much remains unanswered of the dATP preference opposite an abasic site with respect to its functional group and the associated desolvation energies during translesion DNA synthesis. Still, questions revolve around the dynamics adapted for other DNA lesions regarding which biophysical parameters are significantly utilized in the process of translesion DNA synthesis. In this dissertation work I first set out to

31 investigate the following: 1) Which physiochemical forces of the incoming modified

purine nucleotides play an important role during translesion DNA synthesis and normal

DNA synthesis. In, chapter 2, I have used various kinetic methods and modified purines

to address this question 2) How is thymine dimer replicated by T4 DNA polymerase? Is it mediated through a transient abasic site model? I address this question in Chapter 3, in which I examine the efficiency with which T4 DNA polymerase incorporates various non-natural nucleotide analogs opposite thymine dimer, or an abasic site or thymine. In,

Chapter 4, I have specifically used spectroscopic analysis to account for the differences in exonuclease proofreading dynamics of an abasic site and thymine dimer using T4 DNA polymerase. These studies resulted in two publications; one review article and two more publications are in the final stages of preparation.

32

CHAPTER 2

EVALUATING THE BASE STACKING CONTRIBUTIONS OF ALKYLATED AND

UNNATURAL PURINE NUCLEOTIDES DURING TRANSLESION DNA

SYNTHESIS CATALYZED BY BACTERIOPHAGE T4 DNA POLYMERASE

33 2.1. INTRODUCTION

The replication of damaged DNA is considered to be a pro-mutagenic event that can culminate in the development of various diseases, the most notable of which is cancer

(50). A commonly occurring form of DNA damage is an abasic site that can be produced

both non-enzymatically (29) and enzymatically (30) through the action of various DNA

repair enzymes. Although an abasic site is devoid of Watson-Crick hydrogen bonding

potential, most replicative DNA polymerases preferentially incorporate dATP opposite

this lesion. This kinetic phenomenon is commonly referred to as the “A-rule” of

translesion DNA synthesis (37). Indeed, the favorable kinetic evidence obtained in

Berdis lab using bacteriophage T4 DNA polymerase also supports this hypothesis. Our

previous kinetic analysis demonstrated that dATP is incorporated opposite an abasic site

with a ~35-fold greater overall catalytic efficiency compared to dGTP (35). This

difference is caused through enhanced binding affinity for dATP (KD values of 35 μM

versus 130 μM for dATP and dGTP, respectively) as well as through an increase in the

-1 -1 rate constant for polymerization for dATP (kpol values of 0.15 s and 0.023 s for dATP

and dGTP respectively).

Using a series of non-natural indolyl nucleotides, Berdis lab earlier

demonstrated that incorporation of nucleotides can be enhanced through the

enhancements in the π-electron system. Unique among these modified indolyl analogs

-1 are 5-NITP and 5-PhITP which displayed extremely fast kpol values of 126 and 53 s , respectively. In addition, both analogs bind with higher affinity than dATP, having KD values of 18 μM for 5-NITP and 14 μM for 5-PhITP (49). Despite being significantly different with respect to shape and size, both non-natural nucleotides are relatively

34 hydrophobic and rich in π-electron density compared to natural dNTPs. To further validate this model, Berdis lab demonstrated that on replacing π-electron system with a hydrophobic cyclohexane ring, the binding affinity was unaltered but the kpol was altered

by ~315 fold (48). More striking results were observed when the π-electron system was

replaced with an amino group or fluoro group or hydrogen atom. In latter cases, the

binding was highly altered with a poor binding affinity ~20-30 fold higher while the kpol was ~0.20 s-1 which is equivalent to the values of dATP (47). Based on these

observations, Berdis lab proposed that these two biophysical features provide greater

base-stacking capabilities and account for the “A-rule”.

I have extended my studies towards understanding the extent of base stacking and

hydrophobicity influence on promoting and propagating genetic errors during translesion

DNA synthesis by evaluating the commonly formed alklyated purine nucleotides that

commonly arises during the exposure to various alkylating agents that include methyl

methane sulfonate (51), cyclosporamide (52), and mechlorethamine (53). These alkylating agents can also lead to the formation of abasic site by reacting with N7 group

of purines in template that can lead to the hydrolysis of a N-glycosidic bond that occurs

either nonenzymatically (29) or via the action of various DNA gycolysases (30). Thus

far, various reports evaluating the effects of numerous DNA damaging agents are focused

on understanding promutagenic DNA synthesis caused by misreplication of the modified

templating strand. Since the alkylating agents can potentially alter the biophysical

features of the free nucleotides in the biological pool, it could act in concert along with

the alterations in the templating information to synergistically decrease polymerization

fidelity and cause a substantially higher frequency of mutagenesis. A potentially more

35 devastating complication by this process is the risk of developing a secondary cancer

caused by the cytotoxic effects of these DNA-damaging agents (54).

In this current study, I have evaluated the hypothesis that modifications to the

templating nucleobase and incoming nucleotide can lead to a synergistic decrease in

polymerase fidelity. I propose that any alteration in π-electron system or hydrophobicity

of the natural purine by the inappropriate modifications and or by the chemotherapeutic

agents advertently enhances the misincorporation event and potentially leads to more

error propagation during translesion DNA synthesis. I propose that kpol and kpol/KD values for incorporation of these nucleotides should be influenced by the enhancement in the base-stacking and hydrophobic features while alterations in the functional groups at

N7 and N6 of dATP and N7, O6 and N2 of dGTP should perturb the binding affinity. In addition, extension beyond an abasic site will also be influenced through the enhanced base-stacking contributions of the incorporated nucleotide. To test this hypothesis and determine how alterations in the functional groups alter the translesion DNA synthesis, I performed an in-depth kinetic study of the incorporation of modified purine nucleotides opposite an abasic site. To further understand the influence of hydrophobicity and base- stacking capacity on misreplication opposite templating nucleobases, I have performed additional kinetic analysis to distinguish the features of nucleotides that play a significant role during the misreplication opposite templating nucleobases. The work presented in this chapter resulted in a publication in Biochemistry.

36 2.2 MATERIALS AND METHODS

2.2.1 Purification of gp43 DNA polymerase and DNA substrates.

The exonuclease-deficient mutant of gp43 (Asp-219 to Ala mutation) (gift of

S.J. Benkovic, The Pennsylvania State University) was purified and quantified as previously described (55). All oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an abasic site, were synthesized by Operon

Technologies (Alameda, CA). Single-stranded and double-stranded duplex DNA were purified and quantified as described previously (5). The Duplex DNA templates used in this study is in Table 2.1.

Table 2.1 Duplex DNA substrate used in this study

13/20mer 5’-TCGCAGCCGTCCA-3’ 3’-AGCGTCGGCAGGT-x-CCCAAA-5’ x = SP or T or C or A or G

2.2.2 Modified purine nucleotides

The unlabeled modified nucleotides, including 2-APTP, dITP, 6-Cl-dATP, 6-Cl-2-

APTP, 7-deaza-dATP, N2-methyl-dGTP, O6-methyl dGTP, and N6-methyl-dATP (Figure

2.1), were purchased from Trilink BioTechnologies (San Diego, CA) while 7-deaza- dGTP (Figure 2.1) was obtained from Sigma in greater than 99% purity.

37 Figure 2. 1 Chemical structures of modified nucleotides used in this study

O H3C H3C NH O O 2 NH N N NH N N N N NH N N N N NH2 N N N N N NH2 N N N N H R R R R dATP dGTP 6 R N -Me-dATP O6-Me-dGTP N2-Me-dGTP

O Cl Cl

N N N N NH N N N

N N N N N N N NH2 N NH2 R R 6-Cl-dATP R R dITP 6-Cl-2-APTP 2-APTP

NH2 O O O O N NH -O P O P O P O R= O N N N O- O- O- N NH2 R R OH 7-Deaza-dATP 7-Deaza-dGTP

2.2.3 Radiolabeling 5’ends of DNA substrates.

DNA substrates used in this work were radiolabeled prior to their use in enzyme

reactions. Labeling was carried out using T4 polynucleotide kinase in buffer containing

70 mM Tris (pH 9.5), 10 mM MgCl2, 2.5 mM DTT, 2.5% glycerol, 1 mM spermidine,

and 0.1 mM EDTA. [γ-32P]ATP was used as the source of radioactive phosphate, and depending on the remaining specific activity of the ATP and the amount of DNA to be used in the reactions, 3 to 7 % of the DNA substrate was labeled. A typical labeling reaction consisted of buffer, PNK (1 μL), 25 pmol [γ-32P] ATP, 500 pmol 13/20mer

DNA, and dH2O to bring the volume to 100 μL. Since DNA was labeled after

purification of duplexes, the template strand was 5’-labeled in addition to the primer

(substrate) strand. Thus, the template strand is also visible after electrophoresis and

PhosphorImaging. This then serves as a positive control to ensure loading efficiency.

38 The labeling reaction is carried out for 30 minutes at 37oC, followed by heat inactivation of the kinase (10 minutes at 65oC). The labeling mixtures were then slow cooled to room temperature prior to use in enzymatic reactions.

2.2.4 Determinatiion of kinetic rate and dissociation constants for dXTP incorporation opposite an abasic site.

Assays were performed adapting single turnover conditions. 500nM DNA is preincubated with various concentrations (0-500 μM) of nucleotides along with 10 mM

Mg2+. The reaction is initiated with 1 μM gp43 exo-. Prior to initiation of each reaction, microcentrifuge tubes containing 5 μL 200 mM EDTA were prepared for each time point to be examined in these discontinuous assays. After a reaction was initiated, a 5 μL aliquot was removed at each time point and added to an EDTA quench tube, chelating the metal ions and preventing further DNA polymerase activity.

Unless otherwise noted, reactions carried out at time intervals greater than or equal to 4 seconds were done “manually” at the benchtop. The volume of each reaction was 50

μL. A reaction mix containing buffer and metal was prepared (25 mM Tris-OAc, pH 7.5,

150 mM KOAc, 10 mM 2-mercaptoethanol, 10 mM Mg(OAC)2) from concentrated stock solutions. Enzyme (DNA polymerase) and DNA substrate were pre-incubated in this solution, and the reaction was initiated by the addition of dNTPs (10-500 μM). In some instances, these experiments were performed using rapid-quench instrument as previously described with identical conditions (35). Quenched samples were diluted 1:1 with sequencing gel load buffer and the products were analyzed for product formation by 20% denaturing polyacrylamide gel electrophoresis. After the electrophoresis, polyacrylamide

39 gels were transferred to a plastic film backing for support and covered with plastic wrap

on their exposed side. The Phosphor screens were placed on the areas at the location of

the radioactive bands of DNA which was verified using a Geiger counter. The screens

were exposed to the gel from 2-10 hours. The gels were then scanned, producing

quantitatively accurate digital images of the labeled oligonucleotides in the gel.

Intensities from substrates and products were quanitified using Packard PhosphorImager

using the Optiquant Software. The values were normalized by summing all the primer

and product radioactivity in an individual lane. The ratio of each product intensity to total

label intensity (substrate and products) was corrected for background by subtraction of

this ratio as determined in the “zero” lane, which contained no enzyme. The advantage of

this approach was that it controls for variability in loading of DNA in each lane. The rate

constants (kobs) of each reaction were determined by fitting the values described by equation 2.1

y = A* (1-e-kt) + C (2.1)

where A is the burst amplitude, k is the first-order rate constant, t is the time, and C is a defined constant. All reactions were performed in triplicate and the averaged rates for each set of reaction was then fit to Michaelis-Menten described by equation 2.2

kobs = kpol [dXTP]/KD + [dXTP] (2.2)

40 where kobs is the apparent first-order rate constant, kpol is the maximal polymerization rate

constant, KD is the kinetic dissociation constant for dXTP, and [dXTP] is the concentration of nucleotide substrate (Figure 2.2 ).

Figure 2.2 Kinetic scheme of DNA polymerase activity.

dNTP Step 2 E:DNAn E:DNAn:dNTP kpol Step 7 KD Step 1 E':DNAn:dNTP

E + DNAn Step 3

E':DNA :PP PP n+1 i Step 6 i

E:DNAn+1 E:DNAn+1:PPi Step 4 Step 5

2.2.5 Calculations of modified nucleotides biophysical features..

Surface areas (Å2), dipole moment (D), and solvation energy (kcal/mol) for each

nucleobase were calculated using Spartan ’04 software.

2.2.6 Extension beyond an abasic site mispair

Single turnover conditions were employed to measure the rates of extension

beyond a dXMP:abasic site mispair. Gp43 exo- (1 μM) was incubated with 500 nM

DNA (13/20SP-mer) in assay buffer containing EDTA (100μM) and mixed with 100 mM

dXTP and 10 mM Mg(OAC)2. After ~60 s incubation, 900 μM dGTP (the next correct

41 dNTP for the next three positions) were added. Aliquots of the reactions were quenched with 500 mM EDTA at variable times (5-900 s) and analyzed as described above.

42 2.3 RESULTS AND DISCUSSION

2.3.1 Enzymatic incorporation of alkylated purines opposite an abasic site.

The main focus of study is to understand molecular mechanism for the misreplication opposite an abasic site, a DNA lesion that can culminate from the alkylation of purines and pyrimidines (51, 53). Various laboratories including our lab have shown that DNA polymerases preferentially incorporate dATP and subsequently extend beyond the formed mispair to propagate a potential genomic error (56, 57). To further explore how aberrant modifications of purines exacerbate the promutagenic DNA synthesis through the enhancement in the incorporation and extension beyond the mispair by the DNA polymerases, single turnover kinetics analysis with various altered purine nucleotides were performed to determine the features that enhance the binding, polymerization and extension beyond an abasic site.

First, I examined whether subtle modifications to dATP or dGTP that commonly arise during alkylation process can influence the dynamics of misincorporation opposite an abasic site. The first analogue tested is N6-methyl-dATP, a modified purine that arises

through simple alkylation at the N6-position of adenine (Figure 2.1). The observed rate

constants for individual nucleotide concentration are plotted as a function of time yields a

hyperbolic curve (Figure 2.3A). Plotting the observed rate constants as a function of

nucleotide concentration yields a hyperbolic curve (Figure 2.3B). A KD of 190 +/- 45

-1 -1 -1 μM, a kpol of 5.6 +/- 0.6 s and a kpol/KD of 28 420 M s is obtained from the plot. The

7-fold greater catalytic efficiency for N6-methyl-dATP compared to dATP (28420 M-1 s-1 versus 4300 M-1 s-1 respectively) (shown in Table 2.2) indicates that the modified

nucleotide is more promutagenic than the parental nucleotide.

43 Figure 2.3 Dependency of N6-methyl-dATP concentration on the observed rate constant in primer elongation as measured using single-turnover conditions. (A) Dependency of the apparent burst rate constant on the N6-methyl-dATP concentration as measured under single turnover conditions. Assays were performed - 6 using 1 μM gp43 exo , 250 nM 13/20 SP, 10 mM Mg(OAc)2 and N -methyl-dATP in variable concentrations, 25 μM ( ), 50 μM (□),100 μM (◊),250 μM (+) and 500 μM (o). The solid lines represent the fit of the data to a single exponential. (B) The observed rate constants for incorporation ( ) were plotted against N6-methyl-dATP concentration and fit to the Michaelis-Menten equation to determine values corresponding to KD and kpol.

A 250

200

150

100 14mer [nM]

50

0 0123456 Time (seconds)

B 5

4 )

1 3 - (s

obs 2 k

1

0 0 100 200 300 400 500 600 6 N -methyl-dATP [μM]

The enhanced kinetic behavior for N6-methyl-dATP does not arise from an increase in

6 binding affinity as the KD of 190 μM for N -methyl-dATP is ~5 fold higher than the KD

-1 6 of 35 μM for dATP. Instead, the kpol value of 5.6 s for N -methyl-dATP is ~ 40- fold

44 -1 faster than that for dATP (0.15 s ). This dramatic enhancement in the kpol value suggests

that increasing the hydrophobicity and/or overall size of the nucleotide facilitates the

conformational change step that precedes phosphoryl transfer.

I next evaluated whether alkylation of dGTP also increases their promutagenic

potential. dGTP is prone for alkylation modification at O6 and N2 position. O6-methyl-

dGTP and N2-methyl-dGTP were tested opposite an abasic site since alkylation at either

position leads to an overall increase in size and hydrophobicity compared to those of

dGTP (Table 2.2). The catalytic efficiency for O6-methyl-dGTP is ~30-fold greater

compared to that of the incorporation of dGTP (compare 5400 M-1 s-1 with 180 M-1 s-1, respectively). In this aspect, alkylation at the O6 position of dGTP leads to an effect

6 similar to that observed with N -methyl-dATP, i.e., a significant increase in the kpol value

concomitant with decreased binding affinity. In fact, it is striking that the 40-fold faster

6 -1 -1 kpol value with O -methyl-dGTP versus dGTP (compare 0.98 s with 0.023 s ,

6 respectively) is identical to the 40-fold enhancement in kpol values between N -methyl-

dATP and dATP.

The ~3 fold higher catalytic efficiency for N2-methyl-dGTP compared to dGTP

also indicates that alkylation increases the potential for promutagenic synthesis opposite

an abasic site. The enhanced catalytic efficiency is again caused by an increased kpol value rather than an influence on binding affinity. At face value, the increased kpol value

coincides with changes associated with solvation energies between modified and

-1 unmodified nucleobases. However this explanation is incomplete since the kpol of 0.11 s measured with N2-methyl-dGTP is ~ 9-fold slower than that to a relative change in

Gibb’s free energy (∆∆G) of 1.3 kcal/mol and is significantly less that the 2.3 kcal/mol

45 difference in solvation energies between the two analogues (Table 2.2). Similar

observations exist comparing alkylated nucleotides with their unmodified counterparts as

the change in (∆∆G) associated with increases in the kpol values. This analysis clearly

indicates that other biophysical parameters must also contribute to account for the energetic differences. I propose that differences in the aromaticity of the nucleotides as manifest in the different tautomeric forms of N2-methyl-dGTP versus O6-methyl-dGTP

influence their incorporation opposite the non-instructional lesion. This prediction is reasonable since changes in the tautomeric form are known to influence the base-stacking capacity of a nucleotide(58, 59).

I tested this hypothesis by measuring the kinetic parameters for dITP (Figure 2.1), a

nucleotide formed via the deamination of dATP. If hydrophobicity alone dictates

incorporation efficiency opposite an abasic site, then the resulting ~5 kcal/mol decrease

in solvation energy associated with this modification should enhance the incorporation of

dITP opposite the lesion. Contrary to this prediction, I find that dITP is incorporated

-1 -1 very poorly opposite an abasic site with a kpol/KD value of 430 M s . In fact, the low kpol

-1 of 0.044 s and relatively high KD of 103 μM are nearly identical to those for dGTP (kpol

-1 = 0.023 s and KD = 130 μM). Indeed, the striking similarity in the kinetic behavior of dITP and dGTP coincides with the similarities in tautomeric form. Collectively, these data also indicate that the aromatic nature of the nucleotide in addition to its hydrophobicity plays a significant role during translesion DNA synthesis to enhance the promutagenic replication of an abasic site. At the molecular level, this enhancement is achieved through increases in the kpol value that reflect the enhanced base-stacking

capabilities of the nucleotide. On the other hand, binding affinity appears to be adversely

46 affected by any increase in the hydrophobic nature of the incoming nucleotide. One

possibility is that the functional groups on dATP that provide hydrogen-bonding

interactions are in fact essential for achieving optimal binding in the absence of templating information.

To further investigate the role of hydrophobicity and aromatic interactions during translesion DNA synthesis, various unnatural purine triphosphates such as 7-deaza- dATP, 7-deaza-dGTP, 6-Cl-PTP, and 6-Cl-PTP (Figure 2.1) were analyzed opposite an abasic site. The corresponding KD and kpol values for this series of purine analogues are

summarized in Table 2.1. In comparison, modifications and/or atomic substitutions that

increase the hydrophobicity of a nucleotide generally increase the rate constant for

incorporation while reducing binding affinity opposite an abasic site. 7-deaza-dATP

-1 provides a clear example of this kinetic phenomenon as the kpol value of 1.5 s is 10-fold

-1 faster than that of 0.15s for dATP while the KD value of 200 μM is 6-fold higher than the KD of 35 μM for dATP. Similar observations are obtained when the kinetic

parameters for 7-deaza-dGTP are compared with those for dGTP.

Altering the hydrogen-bonding groups at C6 position of dATP with a non-

hydrogen-bonding group does not substantially enhance the dynamics of translesion

DNA synthesis. The case of 6-Cl-PTP shows only a modest 2-fold increase in kpol despite

a 2.81 kcal/mol reduction in solvation energy. The same modification of dGTP yields a

-1 larger effect as the kpol of 0.12 s measured for 6-Cl-2-APTP is 6-fold faster than that of

0.023 s-1 reported for dGTP. However, this increase is most likely caused by a

combination of a change in hydrophobicity and change in tautomeric form to resemble

dATP, the preferred natural nucleotide.

47 Among the nucleotides tested, only 6-Cl-PTP and 6-Cl-2-APTP displayed lowest binding affinities. As argued above, this result suggests that the 6-position of a purine may play an important role in the binding of natural and unnatural nucleotides during translesion DNA synthesis. This argument is well supported by the high KD value of 180

μM measured for 2-APTP, a constitutional analogue of dATP in which the N6 exocyclic amine is permutated to the 2-position. Although this permutation has a strong effect on

-1 binding affinity, the influence on kpol is minimal since the value of 0.23 s is nearly identical, within error, to that of 0.15s-1 for dATP. The similarity in tautomeric form and solvation energies between 2-APTP and dATP again correlated with the identical kpol values.

48 Table 2.1. Summary of kinetic parameters for the incorporation of modified nucleotides opposite an abasic site. a Surface areas (used as an indicator of the relative size of the nucleobase, bsolvation energies and cdipole moments (D), for each nucleobase were calculated using Spartan '04 software.d The tautomeric form refers to whether the purine analogue has the same tautomeric form as dATP or dGTP.e Values taken from (35).f Values taken from (45).g Values taken from ref(49).h NA = not applicable

-1 a b c d dXTP KD [μM] kpol (s ) kpol/KD Surface area Solvation energy Dipole moment Tautomeric [M-1 s-1] (Å2) (kcal/mol) (Debye) form

dATPe 35 +/- 5 0.15 +/- 0.001 4300 143.0 -19.258 2.38 dATP dGTPe 130 +/-5 0.023 +/- 0.005 180 152.5 -26.009 7.18 dGTP

N6-methyl-dATP 190+/-45 5.6 +/- 0.6 29500 165.0 -16.322 2.16 dATP O6-methyl-dGTP 181 +/-35 0.980 +/- 0.008 5400 174.5 -19.998 2.94 dATP N2-methyl-dGTP 245 +/-68 0.12 +/- 0.02 490 173.1 -22.306 7.54 dGTP 49 dITP 103 +/- 14 0.044 +/- 0.003 430 139.0 -21.790 5.63 dGTP

7-deaza-dATP 197 +/- 40 1.4 +/- 0.1 7100 148.7 -17.846 3.64 dATP 7-deaza-dGTP 215 +/- 91 0.11 +/- 0.02 500 158.2 -24.247 4.95 dGTP

6-Cl-PTP 83 +/- 16 0.28 +/- 0.02 3380 145.2 -16.449 4.99 dATP 6-Cl-2-APTP 71 +/- 20 0.12 +/- 0.01 1700 158.7 -19.063 4.83 dATP 2-APTP 180 +/- 23 0.23 +/- 0.02 1300 143.2 -19.142 3.10 dATP

5-NITPf 18 +/-3 126 +/- 7 7 000 000 171.4 -7.381 7.81 NAh 5-PhITPg 14 +/-3 53 +/- 4 3 800 000 223.2 -5.532 3.31 NAh

2.3.2. Extension studies beyond an abasic site

Previously, Berdis lab has shown that gp43 exo- extends beyond an abasic site when dAMP or dGMP is placed opposite the abasic site (35, 59). The dynamics of this process are proposed to reflect the positioning of dATP or dGTP in an interhelical conformation that likely reflects their enhanced base-stacking contributions compared to those of pyrimidines (60, 61). This model leads to a testable prediction: modified purines that possess enhanced base-stacking capabilities should be elongated more easily than their unmodified counterparts. The biological ramifications of this model are obvious since the ability of a modified nucleotide to be elongated predicts a higher promutagenic potential. I have tested this hypothesis with the nucleotides that are considered in these studies using the experimental protocol outlined in Figure 2.4 that monitors the ability of gp43 exo- to extend beyond the various modified purines.

Figure 2.4. Experimental paradigm used to measure the insertion and extension beyond an abasic site.

50 Figure 2.5. Gel electrophoresis analysis showing the extension beyond an abasic site mispair. Gp43exo- (1 μM) and 5’-labeled 13/20SP-mer (500 nM) were preincubated, mixed with 50 μM of dXTP to initiate the reaction. After 2 minutes, an aliquot of the reaction was quenched with 200 mM EDTA (denoted as Inc) to measure insertion opposite the lesion. 900 μM dGTP was then added and aliquots of the reaction were quenched with 200 mM EDTA at 60 sec.

Table 2.3. Summary of kinetic rate constants for extension beyond an abasic site catalyzed by gp43exo- . .

-1 dXTP kext (s ) Solvation Energy Tautomeric Form (kcal/mol) “dATP-like” Analogues dATP 0.25 +/- 0.01 -19.258 A 7-Deaza dATP 0.32 +/- 0.03 -17.846 A N6-Methyl dATP 0.71 +/- 0.04 -16.322 A O6-Methyl dGTP 0.092 +/- 0.009 -22.306 A 6-Cl-PTP 0.031 +/- 0.003 -16.449 A 6-Cl-2-APTP 0.033 +/- 0.004 -19.063 A 2-APTP ND -19.142 A

“dGTP-like” Analogues dGTP 0.005 +/- 0.001 -26.009 G N2-Methyl dGTP ND -19.998 G dITP ND -21.790 G 7-Deaza dGTP ND -24.247 G

51 The gel electrophoresis data is represented in Figure 2.5 and the corresponding

extension rate constants are summarized in Table 2.3 The analysis reveals that most

hydrophobic nucleotides that resemble tautomeric nature of dATP are easily elongated.

In contrast, analogues that are more hydrophilic and/or that resemble tautomeric nature of dGTP are more refractory to elongation. The extension kinetics of nucleotides including

N6-methyl-dATP and 7-deaza-dATP are ~1.5-3 fold faster than that of dATP. This

enhancement coincides with their predicted favorable base-stacking interactions opposite

an abasic site.

It is important to emphasize that the increase in base-stacking capacity depends

upon the hydrophobic and aromatic nature of the nucleotide. This is apparent by

examining the ability of gp43 exo- to elongate beyond N2-methyl-dGTP, dITP and N7-

deaza-dGTP. First, alkylation at the N2-position of dGTP increases its hydrophobicity

and this simple increment does not lead to an enhancement in primer elongation. Second, reducing the solvation energy or increasing the hydrophobicity by removing the N2- functional group or N7 heterocyclic nitrogen group of dGTP also does not lead to extension. This data suggest that the lack in extension is arguably caused by their weaker base-stacking capabilities that reflect reductions in their aromatic nature. Finally, the

6 influence of aromaticity is also evident in the ~20-fold increase in kext for O -methyl-

dGTP that results in a change in tautomeric form caused by alkylation at the O6-position.

Among the nucleotides tested, 2-APTP is the lone exception to this proposed model.

Despite being similar to dATP with respect to tautomeric form and hydrophobicity, this

analogue is not extended when paired opposite an abasic site. Although the molecular

reason for this phenomenon is currently unknown, one possibility is that removal of a

52 functional group at the C6-position prevents contacts between the minor groove of DNA

and the DNA polymerase which are important for polymerase translocation (62, 63).

Consistent with this mechanism is the fact that 6-Cl-PTP is elongated ~8-fold slower compared to dATP. These latter cases suggest that perturbations to the exocyclic amino group may influence the kinetics of elongation. However, the collective data set demonstrates that the base-stacking properties of an incoming nucleotide increase its promutagenic potential by directly influencing incorporation and extension beyond an abasic site.

2.3.3. Enzymatic incorporation of modified purine nucleotides opposite templating DNA

I next evaluated whether the enhanced base-stacking capacity of these modified nucleotides would enhance the misincorporation opposite templating nucleobases. This was initially tested by measuring KD, kpol, and kpol/KD values for this series of purine

analogues opposite thymine and cytosine (Table 2.3), which are predicted to be their

complementary partners. The selectivity factor was calculated which is the ratio of

kpol/KD for nucleotide incorporation opposite its predicted complementary partner versus

its noncomplementary partner. This analysis allows us to classify the modified

nucleotides into three distinct categories: those with high SF values of >103, those with

low SF values of <103, and those with no selectivity (SF values of ~1). In addition, I

have provided a plot of the kpol/KD values for each nucleotide as a function of templating

nucleobases (Figure 2.6). This analysis is important since it indicates whether the SF

values for various nucleotides are caused by an enhancement in the misreplication of a templating base or through diminution in incorporation opposite its predicted “correct” pairing partner or both. Distinguishing among these possibilities is important to

53 accurately interpret how DNA synthesis is perturbed as a consequence of the nucleotide modifications.

Table 2.3. Kinetic parameters of modified nucleotides incorporation opposite thymine and cytosine. a Values taken from (35) . b Estimates were calculated from the linear portion of the Michaelis-Menten plot.

Thymine Cytosine

-1 -1 dNTP KD [μM] kpol (s ) kpol/KD KD [μM] kpol (s ) kpol/KD [M-1 s-1] [M-1 s-1] dATPa 10 +/- 0.5 100 +/- 10 10000000 ND ND <200b dGTP 500 +/- 200 0.04 +/- 0.01 75 5 +/- 2 47 +/- 4 9400000

N6-methyl-dATP 22+/-13 82 +/- 13 4000000 ND ND <40b

O6-methyl-dGTP 465 +/- 190 115 +/- 27 247310 116 +/- 34 20.3 +/- 2 175000

N2-methyl-dGTP 466 +/- 177 0.07 +/- 0.01 150 60 +/- 35 1.7 +/- 0.4 28330 dITP 70 +/- 19 0.080 +/- 0.006 1140 7 +/- 5 49 +/- 10 7000000

7-deaza-dATP 7 +/- 2 80 +/- 9 11430000 270 +/- 90 9 +/- 2 33300

7-deaza-dGTP 850 +/- 400 0.14 +/- 0.04 164 2.4 +/- 0.7 57 +/- 4 23750000

2-APTP 25 +/- 11 119 +/- 20 4760000 90 +/- 38 1.6 +/- 0.2 18100

6-Cl-PTP 25 +/- 5 13 +/- 1 520000 395 +/- 85 0.040 +/- 0.003 105

54 Figure 2.6. Graphical representation of modified nucleotides insertion of opposite thymine or cytosine. (A) Representation of the Selectivity factor (SF) of each modified nucleotide for incorporation opposite DNA containing thymine or cytosine. SF is calculated by (kpol/KD)correct/(kpol/KD)incorrect. Modified nucleotides are classified as those possessing high SF values of >103, those with low SF values of<103 and those with no selectivity (SF values of ~1). (B) kpol/KD values of each nucleotide as a function of templating nucleobase. Red bars represent incorporation opposite thymine, while black bars represent incorporation opposite cytosine.

Using this approach, I find that both unmodified nucleotides dATP and dGTP

are very selective since their calculated SF values are >104. These high SF values are

intuitively obvious since each nucleotide is predicted to interact exclusively with its

complementary partner due to the combination of hydrogen-bonding interactions and

55 steric constraints. In addition, these features are proposed to hinder their misincorporation

opposite their noncognate partners. In this regard, it is surprising that certain analogues

such as 7-deaza-dATP and N2-methyl-dGTP, which have minimal perturbations toward

steric fit and hydrogen-bonding interactions, have significantly lower SF values

compared to their unmodified counterparts. This is interesting since both analogues are

also incorporated opposite an abasic site with a relatively high efficiency. These

coincidences suggest that simply increasing the hydrophobicity of a nucleotide will cause

a unilateral decrease in fidelity. Indeed, this allows us to describe the behaviors of 7- deaza-dATP as the low SF value of 340 is caused by a significant increase in the misincorporation opposite thymine, which is left unperturbed. However, a universal

“hydrophobic effect” is unlikely since the low SF value of 190 for N2-methyl-dGTP is not

caused by an increase for misinsertion opposite thymine. Instead, the reduced fidelity is

caused by a surprisingly large decrease in incorporation opposite cytosine, its predicted

complementary partner.

The best example arguing against a hydrophobic effect causing reduced fidelity in

the presence of templating information is exhibited by N6-methyl-dATP. This highly

promutagenic nucleotide displays the highest catalytic efficiency for incorporation opposite an abasic site. It is remarkable to find that N6-methyl-dATP maintains such an

exquisite selectivity for incorporation opposite thymine versus cytosine. In fact, the SF

value for N6-methyl-dATP is actually 2-fold higher than that for dATP (compare 114,300

versus 50,000, respectively). This feature is not unique to N6-methyl-dATP as similar

trends are also observed with other hydrophobic analogues such as 7-deaza-dGTP and 6-

Cl-dATP which maintain high SF values of >103.

56 One nucleotide that bears special emphasis is O6-methyl-dGTP as this nucleotide

owns the distinction of being the most promutagenic nucleotide identified in this study.

With respect to translesion DNA synthesis, this nucleotide is effectively incorporated

opposite an abasic site with a relatively high catalytic efficiency of 5400 M-1s-1 (Table

2.1). In addition, O6-methyl-dGTP is incorporated opposite thymine and cytosine with a

low SF value of ~1, which indicates complete promiscuity for incorporation opposite pyrimidines. It is quite interesting that the similar catalytic efficiencies of 247,310 and

175,000 M-1s-1 for incorporation opposite thymine and cytosine, respectively, occur through differential perturbations in the measured KD and kpol values. On one hand, the

-1 6 fast kpol of 116 s for incorporation opposite thymine likely reflects the fact that O -

methyl-dGTP has the same tautomeric form as dATP. This interpretation would support

-1 6 the fast kpol value of ~1 s measured for the incorporation of O -methyl-dGTP opposite

an abasic site. However, the binding affinity of O6-methyl-dGTP opposite thymine is

dramatically reduced as manifest in the high KD value of 465 μM. This suggests that ground-state binding opposite a templating base is influenced predominantly by hydrogen-bonding and/or steric-fit constraints rather than by base-stacking interactions.

I performed additional experiments measuring the incorporation of all modified purine triphosphates opposite noncognate partners, adenine and guanine. The gel electrophoresis data is provided in Figure 2.7 and the rate constants are summarized in

Table 2.5. All of the modified purines are poorly incorporated opposite adenine or

guanine as they possess estimated catalytic efficiencies of less than 10 M-1 s-1. In fact, the rate constant for the incorporation of nearly all analogues is <0.01 s-1 even at

nucleotide concentrations greater than 300 μM. In this regard, it is surprising that dATP

57 Figure 2.7 Gel electrophoresis analysis of modified purines incorporation opposite template containing adenine or guanine. Gp43exo- (1 μM) and 5’-labeled 13/20A-mer or 13/20G-mer (500 nM) were preincubated, mixed with 300 μM of dXTP to initiate the reaction. After 4 minutes, an aliquot of the reaction was quenched with 200 mM EDTA to measure insertion opposite the lesion. In the representative gel, only dCTP opposite 13/20G-mer and dTTP opposite 13/20A-mer shows 100% conversion to final product (14mer). All other nucleotides show reductions in the amplitude in product formation.

Opposite 13/20 A Opposite 13/20 G

14-mer 13-mer

o P P P P P P r P P P P P P P P P P T T T T P P P P P P e T T T T P T T T T T T TP T T T T ero T T I T T C G I T T Z G G P T Z C A G P P d - A G G G d - G d dA dA d d P d d d A d - d d d dA d A - - d - d - d d e- Cl A - - Cl - - e e- e e - - a a e 2 - a 6 a M 6 2 z z M z z - -M M a M 6 6 - a - -M a 2 6 6 - a e 2 e N O N N D De O N De - - D - - 7 7 7 7

Table 2.5 Summary of kinetic rate constants for incorporation of modified purine nucleotides opposite adenine and guanine.

Adenine Guanine

-1 -1 dNTP kobs (s ) kobs (s )

dATP 0.021 +/- 0.002 0.007 +/- 0.001 dGTP -ND- 0.031 +/- 0.001

N6-methyl-dATP 0.0020 +/- 0.001 0.009 +/- 0.01 O6-methyl-dGTP 0.005 +/- 0.001 0.004 +/- 0.001 N2-methyl-dGTP -ND- 0.003 +/- 0.001 dITP 0.0010 +/- 0.00002 0.003 +/- 0.001

7-deaza-dATP 0.0010 +/- 0.0003 0.0010 +/- 0.002 7-deaza-dGTP -ND- -ND- 2-APTP 0.005 +/- 0.001 0.003 +/- 0.001 6-Cl-PTP 0.002 +/- 0.002 0.003 +/- 0.001

58 is incorporated opposite adenine with a relatively high catalytic efficiency of 420 M-1 s-1.

A similar phenomenon is observed for the incorporation of dGTP opposite guanine in which the overall catalytic efficiency is estimated to be 100 M-1 s-1.

Collectively, the kinetic data obtained using these modified purine analogues do

not provide evidence for a strong correlation between their hydrophobic and/or aromatic

nature and the ability to form a mispair. In this regard, the dynamics by which a

polymerase misreplicates a templating base appear to be vastly different from the

dynamics for the misincorporation of a nucleotide opposite an abasic site. This

conclusion has several important ramifications. First, differences in the underlying

mechanism by which damaged DNA is misreplicated provide insight regarding why the

mutagenic spectra of closely related DNA-damaging agents are different. For example,

dCTP and dTTP are typically incorporated opposite damaged nucleobases such as O6-

methylguanine and O6-ethylguanine that contain small modifications. This implies that

these lesions are replicated using features associated with classical hydrogen-bonding and

steric-fit constraints similar to an unmodified guanine. However, the introduction of

larger substituent groups at the O6-position of guanine gives rises to a different

mutational spectrum in which dATP or dGTP is preferentially incorporated (64). In this

latter case, the preferential incorporation of dATP argues that larger DNA lesions are

replicated via a “transient abasic site intermediate”. In any event, this information could be used to develop a predictive index for the mutagenic potential of other DNA-

damaging agents. Another practical application is toward the rational design of antiviral

agents that take advantage of low-fidelity replication catalyzed by most viral polymerases

(65, 66). Lower replication fidelity is generally tolerated by many viruses. However, the

59 introduction of too many mutations, referred to as error catastrophe, may cause a loss of

viral fitness and viability. This phenomenon has led to the development of a chemotherapeutic strategy termed “lethal mutagenesis” in which promutagenic

nucleotides are used to increase the mutation frequency of viruses beyond viability to

induce error catastrophe (67).

60 2.1 CONCLUSION

In this work I demonstrated that alkylated purine triphosphates can significantly enhance the promutagenic properties of an abasic site. My kinetic data yield further insights into the molecular mechanism of translesion DNA synthesis. During the replication of an abasic site, we argue that the enzymatic conformational change step (as reflected by the kpol value) is influenced by the hydrophobic and aromatic nature of the incoming nucleotide. This conclusion is consistent with the previously published model derived from characterizing the incorporation of various 5-substituted indolyl nucleoside triphosphates opposite this non-instructional DNA lesion (68). Non-natural nucleotides such as 5-NITP and 5-PhITP are incorporated opposite this non-instructional lesion with incredibly high catalytic efficiencies (>106 M-1 s-1) (45). Both analogues have low affinity constants (KD values <20 μM) coupled with fast incorporation rate constants

-1 (>50s ). The faster kpol values measured with these 5-substituted indolyl nucleotides likely reflect their enhanced base-stacking capabilities that originate from their large π- electron surface area and hydrophobic nature.

The ability of various modified purines to be efficiently elongated has important biological ramifications, especially within the context of replicating non-instructional

DNA lesions such as an abasic site. Simple alklyation of dATP or dGTP enhances the efficiency of translesion DNA synthesis that could cause a synergistic increase in mutagenesis under certain circumstances. The provocative implication of this conclusion is the potential risk of certain chemotherapeutic modalities to induce mutagenesis that may result in an additional prooncogenic event. Indeed, the administration of certain

61 cytotoxic DNA-damaging agents such as temozolomide and cyclophosphamide is

associated with a 10-fold higher risk of developing a secondary cancer (69).

Finally, the dynamics by which these modified purines are misincorporated are highly dependent upon the presence or absence of templating information. On one hand, increasing the hydrophobicity and aromaticity of a nucleotide leads to enhanced efficiency in incorporation and extension beyond an abasic site. However, a much more complex scenario exists during misinsertion opposite templating bases. In general, the catalytic efficiency for misinsertion depends more upon alterations in hydrogen-bonding interactions and shape complementarity as opposed to perturbations in hydrophobicity.

Of course, unambiguous conclusions are difficult to make since modifying the functional group of an incoming nucleotide influences all three biophysical features to varying degrees. This last feature is important as it questions whether a truly universal mechanism of polymerization exists during the replication of normal versus damaged

DNA. The data presented in this chapter suggest that different biophysical rules apply during template-dependent versus template-independent DNA synthesis. Therefore, strict comparisons between the mechanisms of translesion DNA synthesis and correct DNA synthesis may be invalid. This dichotomy may explain the ease with which non-natural nucleotides can be designed to be efficiently incorporated opposite a non-instructional lesion while such efforts have proven more difficult in creating hybrid base pairs composed of natural and unnatural nucleotides.

62

CHAPTER 3

EVALUATING THE BASE STACKING CONTRIBUTIONS DURING BYPASS OF

THYMINE DIMER CATALYZED BY T4 DNA POLYMERASE

63 3.1 INTRODUCTION

We previously demonstrated, using a series of non-natural indolyl nucleotides, that base stacking contributions of the incoming nucleotide are predominantly utilized during incorporation opposite an abasic site by high-fidelity DNA polymerases. However, it is still unclear whether DNA polymerases adopt a similar kind of mechanism to bypass various structurally different DNA lesions that are promoted through UV radiation and oxidizing agents. The major class of photoproducts that results from the absorption of

UV light are the cis,syn cyclobutane dimers and 6-4 products (70). In eukaryotic systems, this lesion is repaired via the nucleotide excision repair pathway or bypassed by pol η in an error-free mechanism (71). Unfortunately, this lesion is also proposed to be bypassed by high fidelity DNA polymerase in the absence of pol η to promote characteristic mutations in dyprimidne sequences (72). For example, squamous cell carcinoma (73) involves mutations in the p53 gene(74) while basal cell carcinoma and melanoma involve mutations in the PATCHED gene and the p16 gene (75-77) respectively.

The replication beyond a thymine dimer by replicative polymerase was first reported with bacteriophage T7 DNA polymerase that has been extensively evaluated through a series of kinetic and structural studies (78, 79). This high-fidelity DNA polymerase is proposed to replicate thymine dimer as a non-instructional lesion rather than as a misinstructional lesion (78). The ability of a DNA polymerase to replicate a thymine dimer as an abasic site is intriguing since the structures of these lesions differ significantly (Figure 3.1).

64 Figure 3.1. Structures of thymine dimer and abasic site.

O O O -O P O

HN NH O O

N N O O O H H

Thymine Dimer Abasic site

This conclusion is based upon nucleotide selectivity studies comparing the ability of the T7 DNA polymerase to incorporate dATP or pyrene triphosphate (dPTP) opposite a thymine dimer versus an abasic site (78). Briefly, the T7 polymerase incorporates dPTP more effectively than dATP opposite an abasic site. The large degree of selectivity of

~58 for dPTP versus dATP was interpreted to reflect the influence of shape- complementarity during the misreplication of the non-instructional DNA lesion (78).

During replication opposite the 3’-T of a thymine dimer, the selectivity for dPTP incorporation versus dATP is ~15-fold. It is clear that the magnitude of this selectivity is less than that of ~58 measured for incorporation opposite an abasic site. However, these data clearly establish that the large, non-natural nucleotide is preferred over its smaller natural counterpart, and this result is consistent with the ability of the polymerase to replicate a thymine dimer as a non-instructional lesion. This model is supported by structural evidence of the T7 exo- DNA polymerase bound at thymine dimer in which the lesion lies outside the helical structure of DNA (79) to create a cavity that functionally resembles an abasic site (Figure 3.2).

65 Figure 3.2. Crystal structure of T7 DNA polymerase with DNA template containing thymine dimer (ref). Pdb : 1SKS

3’OH

Thymine Dimer

These observations led us to question if similar mechanisms are employed by

bacteriophage T4 DNA polymerases during the replication of cis,syn thymine dimer

DNA lesions. Therefore, I performed a thorough kinetic analysis comparing the ability of

the bacteriophage T4 DNA polymerase (gp43) to incorporate various natural and non-

natural nucleotides opposite a thymine dimer versus an abasic site.

Our lab previously demonstrated that non-natural analogs containing significant π-

electron surface areas such as 5-NITP, 5-PhITP, and 5-NapITP are incorporated ~1,000-

fold more efficiently than analogs such as 5-AITP and 5-CHITP that have substantially

smaller π-electron surface areas (49, 80, 81). These results are consistent with a model in which the overall catalytic efficiency of nucleotide incorporation is directed by the base-

stacking capabilities of the incoming nucleotide. In this model, the binding affinity (KD) of the incoming nucleotide is linked with its shape and hydrophobicity while the rate

66 constant for incorporation of these non-natural nucleotides is solely dependent on the presence of π-electron density.

These results prompted us to test the following hypothesis: if gp43 processes a thymine dimer as a transient "abasic site", then analogs such as 5-NITP, 5-PhITP, and 5-

NapITP are expected to be incorporated opposite either lesion with nearly identical kinetic parameters. This report outlines the structure-activity relationships derived from monitoring the incorporation of various 5-modified indolyl nucleotides (Figure 3.3) opposite a thymine dimer. These current studies reveal that gp43 processes a thymine dimer via a mechanism that is similar but not identical to an abasic site. Significant differences in the kinetic parameters are found as a consequence of DNA lesion. This indicate that the mechanisms are not identical for gp43 as previously proposed for the T7

DNA polymerase (78). Kinetic evidence is provided that the conformational change step preceding phosphoryl transfer is rate-limiting for the incorporation of these analogs irrespective of DNA lesion. In addition, notable similarities are detected in the mechanisms of the proofreading of natural nucleotides paired opposite either DNA lesion. However, distinct differences are observed using non-natural nucleotides in which

5-PhIMP can be excised via exonuclease and pyrophosphorolytic activity when placed opposite an abasic site but not when placed opposite a thymine dimer. The collective data set is used to develop a comprehensive model highlighting the similarities and differences in the replication of a non-templating lesion versus a bulky miscoding DNA adduct. The work presented in this chapter resulted in a publication in Biochemistry.

67 3.2 MATERIALS AND METHODS

3.2.1 Protein purification, preparation of DNA substrates and synthesis of non-natural

nucleotides

Wild-type gp43 and the exonuclease-deficient mutant gp43 (D219A) mutant were

purified and quanitified as described previously (55). The oligonucleotide containing a

cis,syn thymine dimer was synthesized by Trilink Biotechnologies (San Diego,CA). All

other oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an

abasic site, were synthesized by Operon Technologies (Alameda, CA). Single-stranded

and double-stranded duplex DNA used in this study (Table 3.1) were purified and

quantified as described previously. The non-natural nucleotides used in this study were

synthesized and purified as described previously (47, 49, 80, 81). (Figure 3.3)

Table 3.1 Duplex DNA substrates used in this study

13/20T=T-mer 5’-TCGCAGCCGGTCC-3’ 3’-AGCGTCGGCCAGGT=TCCAAA-5’ T=T denotes thymine dimer

13/20SP-mer 5’-TCGCAGCCGGTCC-3’ 3’-AGCGTCGGCCAGGSPCCAAA-5’ SP denotes an abasic site

13/20T-mer 5’-TCGCAGCCGGTCC-3’ 3’-AGCGTCGGCCAGGTCCCAAA-5’ T denotes thymine

14A/20T=T-mer 5’-TCGCAGCCGGTCCA-3’ 3’-AGCGTCGGCCAGGT=TCCAAA-5’

14A/20SP-mer 5’-TCGCAGCCGGTCCA-3’ 3’-AGCGTCGGCCAGGSPCCAAA-5’

68 Figure 3.3 Chemical structures of non-natural nucleotides

NH2 O F NH2 +N N N O-

N N N N N N dR dR dR dR dR IndTP 5-FITP dATP 5-AITP 5-NITP

N N N N dR dR dR dR

5-CHITP 5-CEITP 5-PhITP 5-NapITP

N O O O dR dR=-O P O P O P O O 5-AnITP O- O- O- OH

3.2.2 Determination of the kinetic rate and dissociation constants for dXTP incorporation

opposite a thymine Dimer.

In most cases, a rapid quench instrument (KinTek Corporation, Clarence, PA) was

used to monitor the time courses in nucleotide incorporation opposite a thymine dimer.

o Experiments were performed at 25 C in which 250 nM 13/20T=T-mer and variable

concentrations of nucleotide analog (5 – 500 μM) were pre-incubated in assay buffer (25 mM Tris-OAc pH 7.5; 150 mM KOAc, and 10 mM 2-mercaptoethanol) and mixed versus

1 μM gp43 exo- and 10 mM Mg(OAc)2. The reactions were quenched with 500 mM

69 EDTA at variable times (0.005-10 sec). The polymerization reactions were monitored by

analysis of the products on 20% sequencing gels (5). Gel images were obtained from a

Packard PhosphorImager using the OptiQuant software supplied by the manufacturer.

Product formation was quantified by measuring the ratio of 32P-labeled extended and

non-extended primer. The ratios of product formation were corrected for substrate in the

absence of polymerase (zero point). Corrected ratios were then multiplied by the

concentration of primer/template used in each assay to yield total product. Data obtained

for single turnover DNA polymerization assays were fit to equation 3.1. y = A(1-e-kt) + C (3.1)

where A is the burst amplitude, k is the observed rate constant (kobs) in initial product

formation, t is time, and C is a defined constant. Data for the dependency of kobs as a

function of dXTP concentration was fit to the Michaelis-Menten equation (equation 3.2)

to provide values corresponding to kpol and KD

kobs = kpol[dXTP]/(KD + [dXTP]) (3.2)

where kobs is the observed rate constant of the reaction, kpol is the maximal polymerization

rate constant, KD is the kinetic dissociation constant for dXTP, and dXTP is the

concentration of non-natural nucleotide substrate.

3.2.3 Determination of rate limiting step using Acid versus EDTA as the quenching

agent.

Two fifty nanomolar of DNA (13/20T=T) was incubated with KD concentrations of

non-natural nucleotide and 10mM Mg(OAc) 2. The reaction was then initiated with 1 μM

gp43 exo- and quenched with either 500 mM EDTA or 1M HCl at time intervals ranging

70 from 0.1 sec to 10 sec using a rapid quench instrument as described above. After

quenching with 1M HCl, 100 μL of phenol/choloroform/iso-amyl alcohol was added to

extract the DNA polymerase, and the pH of the aqueous phase was neutralized by addition of ~30 μL of 1M Tris/3M NaOH. Product formation was analyzed and

quantified as described above.

3.2.4 Idle-Turnover Measurements

Two fifty nanomolar of DNA (13/20T=T-mer or 13/20SP-mer) was first

preincubated with KD concentrations of 5-PhITP (40 μM) in the presence of 30 μM dATP. Due to the nature of the DNA substrate (Figure 1A), dAMP insertion opposite T at position 13 in the template maintains a usable primer template for the insertion of the non-natural nucleotide opposite the thymine dimer lesion (position 14). In all cases, the reaction was initiated by the addition of 1 μM gp43 exo+. Reactions were quenched with

500 mM EDTA at time frames ranging from 5 to 600 seconds. The quenched samples

were processed and the product formation was analyzed as described (81).

3.2.5 Exonuclease Degradation of Unmodified and Damaged DNA

Exonuclease reactions were performed under single turnover reaction conditions in which

250 nM DNA was preincubated with 10 mM Mg(OAc)2 in assay buffer, and the reaction was

initiated by adding 1 μM gp43 exo+. These studies include monitoring the enzymatic hydrolysis

of the following DNA substrates: 14A/20T=T -mer, 14A/20SP -mer, 13/20T=T -mer, 13/20SP -mer, and 13/20T -mer. In all cases, a rapid quench instrument was used to quench the reactions using

500 mM EDTA at time intervals ranging from 0.003 to 2 sec. Degradation products were

71 analyzed as described above and data points were plotted as initial substrate (13-mer) remaining

as a function of time. Data for each time course were fit to equation 3.3 defining a first order

decay in initial substrate concentration y = Ae-kt + C (3.3)

where A is the amplitude of the burst phase, k is the observed rate constant for product

formation, and C is the end point of the reaction.

3.2.6 Pyrophosphorolysis

Five hundred nanomolar of DNA (14A/20T-mer, 14A/20T=T-mer, 14A/20SP-mer)

- was first incubated with 50 nM of gp43 exo and 10 mM Mg(OAc)2. The reaction was

then initiated with 20 mM of PPi and quenched with 500 mM EDTA at time intervals (5-

300 seconds). The quenched samples were processed as described above and the product

formation was analyzed using protocols similar to that used to monitor the exonuclease

activity of gp43 (81).

The pyrophosphorolytic activity of gp43 using 5-PhITP as the non-natural

nucleotide was performed using a modified procedure. 500 nM of 13/20T=T-mer or

- 13/20SP-mer was incubated with 50 nM of gp43 exo , 10 mM Mg(OAc)2, and 10 μM 5-

PhITP. In this case, the polymerase was allowed to incorporate enzymatically 5-PhITP

opposite the DNA lesion to create the 14-mer. After ~20 minutes (the time required to

achieve >90% conversion of 13-mer to 14-mer), pyrophosphorolysis was initiated with

20 mM of PPi. The reaction was quenched through the addition of 500 mM EDTA at time

intervals (5-300 seconds) and processed as described above.

72 3.3 RESULTS AND DISCUSSION

The series of natural and non-natural nucleotides illustrated in Figure 3.3 were

used to probe for similarities or differences in the ability of gp43 to replicate a thymine

dimer or an abasic site. In either case, single turnover conditions were employed in which

1 μM gp43exo- was added last to a preincubated solution of 50 μM of dXTP and 500 nM

DNA (13/20T=T-mer or13/20SP-mer). Preliminary screening comparing the incorporation

of these dXTPs opposite either DNA lesion is provided in Figure 3.4.

Figure 3.4. Screening of non-natural nucleotides incorporation opposite an abasic site and thymine dimer. 500nM of 13/20SP-mer or 13/20T=T-mer is preincubated with 50 μM of dXTP and 10 mM Mg2+ and the reaction was initiated with 1 μM gp43 exo-. The reaction was quenched at 60sec with 200mM EDTA and analyzed for the product formation as previously described.

o r

e Z dATP IndTP 5-FITP 5-AITP 5-NITP

SP T=T SP T=T SP T=T SP T=T SP T=T 14mer

13mer

5-ChITP 5-CEITP 5-PhITP 5-NapITP

SP T=T SP T=T SP T=T SP T=T

14mer

13mer

In general, significant differences are observed for the incorporation of non-natural

nucleotides opposite either DNA lesion and suggest that gp43exo- does not process the

thymine dimer via the "A-rule". Among the nucleotides analyzed, IndTP, 5-FITP, and 5-

AITP are readily incorporated opposite an abasic site but they are not incorporated

73 opposite the thymine dimer. The most striking difference, however, is that dATP and 5-

NITP are poorly incorporated opposite the thymine dimer. This is noteworthy since

dATP and 5-NITP are efficiently incorporated opposite an abasic (Figure 3.4), and this

result recapitulates previously reported data (35, 45). It should also be noted that of the four natural nucleotides, only dATP showed any incorporation opposite the thymine dimer at a concentration of 500μM (Figure 3.5).

Figure 3.5 Enzymatic incorporation of natural nucleotides opposite thymine dimer. 500nM of 13/20T=T was preincubated with 500μM of either dATP (red) or dGTP (blue) dCTP (green) and dTTP (black) and 10 mM Mg2+ and the reaction was initiated with 1 mM gp43 exo-. The samples were quenched at regular intervals with 200mM EDTA and the rate constants (kobs) were determined described previously.

10

8

6

4

2

0 0 20406080100120140 Time (sec)

However, even at the highest concentration of dATP tested (500 μM), the measured rate

-1 -1 constant of 0.007 sec is ~21-fold slower than the reported kpol value of 0.15 sec measured for dATP incorporation opposite an abasic site (35). In general, the difference

74 in utilization of these analogs suggests that gp43 exo- replicates a thymine dimer via a

different mechanism than that reported for an abasic site (35, 45, 47).

There are, however, instances in which gp43 exo- does incorporate certain non-

natural nucleotides opposite either lesion with comparable efficiencies. As illustrated in

Figure 3.4, indolyl analogs containing large π-electron surface areas such as 5-CEITP, 5-

PhITP, and 5-NapITP are incorporated opposite the thymine dimer almost as effectively

as an abasic site. Consequently, there is an apparent dichotomy in nucleotide utilization

that cannot be rationalized solely through examining these qualitative data. The

underlying reasons for these differences in nucleotide utilization were thoroughly

investigated using a quantitative kinetic approach described below.

3.3.1 Non-natural nucleotides incorporation opposite a thymine dimer.

KD and kpol values were measured for the subset of non-natural nucleotides

incorporated opposite the thymine dimer. All experiments were performed using single

turnover conditions as previously described (35). Representative data provided in Figure

3.6 (A) shows the time courses in 5-NapITP incorporation opposite the thymine dimer.

All time courses were fit to the equation for a single exponential process to define kobs, the rate constant in product formation. As shown in Figure 3.6 (B), the plot of kobs versus

5-NapITP concentration is hyperbolic, and a fit of the data to the Michaelis-Menten

-1 equation yields a kpol value of 6.4 +/- 0.5 sec and a KD value of 13 +/- 3 μM. Identical

analyses were performed with other non-natural nucleotides, and the corresponding KD and kpol values are summarized in Table 3.2.

Inspection of Table 3.2 clearly indicates that analogs such as 5-CEITP, 5 PhITP,

and 5-NapITP are preferentially incorporated opposite a thymine dimer. The catalytic

75 Figure 3.6 Enzymatic incorporation of 5-NapITP opposite thymine dimer. (A) Dependency of the apparent burst rate constant on the concentration 5-NapITP as measured under single turnover conditions. Assays were performed using 1 μM gp43 - exo , 250 nM 13/20 SP, 10 mM Mg(OAc)2 and 5-NapITP in variable concentrations, 5 μM ( ), 10 μM (V), 25 μM (□), 50 μM (+) and 100 μM (S). The solid lines represent the fit of the data to a single exponential. (B) The observed rate constants for incorporation ( ) were plotted against 5-NapITP concentration and fit to the Michaelis- Menten equation to determine values corresponding to KD and kpol.

A 250

200

150

100 14mer [nM]

50

0 00.511.52 Time (Seconds)

B 6 5 )

-1 4 (s 3 obs k 2 1 0 0 20406080100120 5-NapITP [μM]

efficiency (kpol/KD) for these analogs is between 2- and 4-orders of magnitude greater

than that for smaller analogs such as 5-AITP and 5-FITP that lack extensive π-electron

density. It is striking that the overall catalytic efficiency increases as the π-electron

surface area increases. In general, this variation reflects alterations in binding affinity

76 rather than perturbations in polymerization rate constant since the kpol values for 5-

CEITP, 5-PhITP, and 5-NapITP are nearly identical at ~4-6 sec-1. The mechanistic significance of these observations is discussed below.

Table 3.2. Summary of kinetic rate and equilibrium constants measured for the insertion of dATP and 5-substituted indolyl-2’-deoxyriboside triphosphates opposite thymine dimer and abasic site. a ND = Not Determined.bAccurate values could not be determined since the lack of saturation kinetics prohibited the determination of true kpol and KD values. Thus, the reported catalytic efficiencies reflect upper estimates based upon the rate constant (kobs) measured using 500 μM dXTP divided by the highest concentration of nucleotide tested (500 μM).cValues taken from (35) d Values taken from (45) eValues taken from (47) fValues taken from(48) gValues taken from (49) hValues taken from (80)

Thymine Dimer Abasic site

-1 -1 dXTP KD [μM] kpol (s ) kpol/KD KD [μM] kpol (s ) kpol/KD [M-1 sec-1] [M-1 sec-1] dATP ND a 0.007 +/- 0.002 <20 b 35 +/- 5 0.15 +/- 0.01 4,600 c

5-NITP 39 +/- 19 0.042 +/- 0.006 1,100 18 +/- 3 126 +/- 6 7,000,000 d

5-FITP ND 0.030 +/- 0.004 <60 b 152 +/- 41 0.30 +/- 0.03 2,000 e

5-CHITP ND 0.059 +/- 0.004 <120 b 6.2 +/- 1.3 0.46 +/- 0.003 74,200 f

5-CEITP 119 +/- 31 4.4 +/- 0.5 36,980 4.6 +/- 1.0 25.1 +/- 1.5 5,460,000 f

5-PhITP 36 +/- 13 4.4 +/- 0.43 122,000 14 +/- 3 53 +/- 4 3,800,000 g

5-NapITP 13 +/- 3 6.4 +/- 0.5 492,300 10.3 +/- 4.5 27.1 +/- 1.5 2,631,100 h

5-AnITP 31 +/- 19 1.6 +/- 0.5 51,600 27 +/- 7 5.3 +/- 0.4 200,000 h

77 The favorable incorporation of analogs containing large π-electron surface areas

suggests that the thymine dimer can be processed as a non-instructional lesion since

similar data were obtained for their incorporation opposite an abasic site (48, 49, 80).

However, the notable variations in kinetic parameters indicate that the misreplication of

each lesion occurs via a distinct mechanism. One clear example is the striking difference

in binding affinities of these analogs that are dependent upon the nature of the lesion.

Specifically, the KD values of 5-CE-ITP, 5-PhITP, and 5-NapITP are essentially identical

at ~15 μM for incorporation opposite an abasic site (47, 80). During replication of a

thymine dimer, however, these values vary significantly and appear linked with the

overall π-electron surface area of the incoming dXTP. Of these three analogs, 5-NapITP

has the highest binding affinity (KD = 13 μM) which coincides with its large π-electron surface area (273 Å2). In contrast, the smaller π-electron surface area of 5-CE-ITP (183

2 Å ) arguably causes a weaker binding affinity as manifest in the 10-fold higher KD value of 120 μM. These differences are interesting since a correlation between binding affinity and π-electron surface area is not observed during incorporation opposite an abasic site.

Differences are also detected with respect to the kpol values which are again

dependent upon the nature of the lesion. kpol values measured with abasic site containing

DNA vary from 25 to 50 sec-1 while they remain essentially invariant during

incorporation opposite a thymine dimer. The most dramatic effect, however, is that the

rate constants for incorporation are considerably slower opposite the thymine dimer. The magnitude for this effect is largest with 5-PhITP in which the kpol for an abasic site is 12-

fold faster than with the thymine dimer (compare 53 sec-1 versus 4.4 sec-1, respectively).

78 Perhaps the most unique kinetic behavior is again evident when evaluating the kinetic

parameters for 5-NITP. The preliminary data provided in Figure 3.4 reveals that 5-NITP

is poorly incorporated opposite a thymine dimer.

Figure 3.7. Enzymatic incorporation of 5-NITP opposite thymine dimer. (A) Dependency of the apparent burst rate constant on the concentration 5-NITP as measured under single turnover conditions. Assays were performed using 1 μM gp43 - exo , 250 nM 13/20 SP, 10 mM Mg(OAc)2 and 5-NITP in variable concentrations, 5 μM ( ), 10 μM (□), 50 μM (+), 150 μM (c) and 250 μM (d). The solid lines represent the fit of the data to a single exponential. The observed rate constants for incorporation ( ) were plotted against 5-NITP concentration and fit to the Michaelis-Menten equation to determine values corresponding to KD and kpol.

A 120 100 80 60

40 20

0 0 50 100 150 200 250 Time (seconds)

B 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0 50 100 150 200 250 300 5-NITP [μM]

79 However, this low efficiency partially reflects the reaction conditions employed in this

experiment, i.e., low dXTP concentration and a short reaction time of 10 seconds. In fact,

increasing the concentration of 5-NITP and monitoring the reaction at time frames

encompassing 15 to 600 seconds resulted in a dose and time-dependent increase in

product formation [Figure 3.7 (A)]. From these experiments, a KD of 39 +/- 19 μM and a

-1 kpol value of 0.042 +/- 0.006 sec were measured [Figure 3.7(B)]. The KD of 39 μM is

only 2-fold higher than the value of 18 μM measured for an abasic site (45). Thus, the

presence of π-electron density on the incoming nucleotide appears to influence binding

-1 affinity. In contrast, the kpol value of 0.042 sec measured using the thymine dimer is

3,000-fold lower than the value of 126 sec-1 measured for an abasic site (45). This

difference suggests that the presence of π-electron density alone is insufficient to

facilitate the conformational change that limits nucleotide incorporation. In fact, it

appears that the kpol values for incorporation opposite the thymine dimer depend equally

upon contributions from both π-electron density and overall size of the analog. This finding is reminiscent of the shape-complementarity model originally proposed by Kool and co-workers (82).

To test this hypothesis, I next evaluated the kinetic parameters for 5-AnITP, the analog with the largest π-electron surface area, opposite the thymine dimer. It is quite interesting that of all of the non-natural nucleotides tested in this study, only 5-AnITP displays nearly identical kinetic parameters regardless of DNA lesion. For example, similar KD values of 31 and 27 μM are obtained for incorporation opposite a thymine

-1 dimer versus an abasic site, respectively (80). Furthermore, the kpol value of 1.6 sec for

incorporation opposite a thymine dimer is only 3-fold slower than the value of 5.3 sec-1

80 reported using an abasic site (49). While these data are consistent with the aforementioned hypothesis, it should be noted that 5-AnITP also displays unique kinetic parameters for insertion opposite templating nucleobases. Specifically, the KD of 29 μM

for incorporation opposite thymine is not significantly different from that of 31 μM for

-1 incorporation opposite a thymine dimer. Likewise, the kpol of 0.5 sec is only 3-fold

slower than the value of 1.6 sec-1 measured for incorporation opposite a thymine dimer.

This last piece of kinetic data suggests that the thymine dimer may be replicated as

a templating nucleobase rather than as an abasic site. To evaluate this possibility, we provide Table 3.3 which compares the kinetic parameters for these non-natural nucleotides opposite a thymine dimer versus an unmodified thymine. Certain analogs

lacking large π-electron surface areas (5-NITP, 5-FITP, and 5-CH-ITP) are incorporated

far more effectively opposite T compared to the cross linked DNA adduct. However,

analogs such as 5-CE-ITP, 5-PhITP, and 5-NapITP that contain large π-electron surface

areas are incorporated more effectively opposite a thymine dimer compared to thymine.

In these latter instances, the increased efficiency is caused by faster kpol values rather than

through an enhancement in binding affinity.

81 Table 3.3. Summary of kinetic rate and equilibrium constants measured for the incorporation of dATP and 5-substituted indolyl-2’-deoxyriboside triphosphates opposite a thymine dimer or thymine. a ND = Not Determined.b Accurate values could not be determined since the lack of saturation kinetics prohibited the determination of true kpol and KD values. Thus, the reported catalytic efficiencies reflect upper estimates based upon the rate constant (kobs) measured using 500 μM dXTP divided by the highest concentration of nucleotide tested (500 μM).c Values taken from (5). d Values taken from (45). e Values taken from (49). f Values taken from (48) . g Values taken from (80) .

Thymine Dimer Thymine

-1 -1 dXTP KD [μM] kpol (s ) kpol/KD KD [μM] kpol (s ) kpol/KD [M-1 sec-1] [M-1 sec-1]

dATP NDa 0.007 +/- 0.002 <20 b 10 +/- 0.5 100 +/- 10 10,000,000 c

5-NITP 39 +/- 19 0.042 +/- 0.006 1,100 9 +/- 1 0.9 +/- 0.1 100,000 d

5-FITP NDa 0.030 +/- 0.004 <60 b 141 +/- 32 0.040 +/- 0.003 2,900 e

5-CHITP NDa 0.059 +/- 0.004 <120 b 25 +/-10 0.018 +/- 0.005 720 f

5-CEITP 119 +/- 31 4.4 +/- 0.5 36,980 63 +/-12 0.076 +/- 0.005 1,120 f

5-PhITP 36 +/- 13 4.4 +/- 0.4 122,000 25 +/- 7 0.16 +/- 0.01 6,400 f

5-NapITP 13 +/- 3 6.4 +/- 0.5 492,300 16 +/- 8 2.21 +/- 0.4 135,600g

5-AnITP 31 +/- 19 1.6 +/- 0.5 51,600 29 +/- 15 0.53 +/- 0.11 18,600 g

82 Figure 3.8. Proposed models for the enzymatic incorporation of non-natural nucleotides. The first represents the binding of dNTP to the polymerase:DNA complex (KD). After nucleotide binding, the polymerase undergoes a conformational change (kpol) that is required to place the triphosphate moiety in close proximity with the positively charged amino acids as well as to stack the nucleobase portion of the incoming dNTP into the hydrophobic environment of the interior of the duplex DNA. The final stage of the catalytic cycle is phosphoryl transfer step that is required for elongation of the primer strand (kchem). Panels B-E provides computer-generated models for DNA containing an abasic site (B-C) or a thymine dimer (D-E). All models were constructed using Spartan '04 software. (B) Computer generated model for the structure of DNA containing an abasic site. (C) 5-Phenyl-indole-deoxyribose monophosphate paired opposite an abasic site in which the non-natural nucleobase is placed in an intrahelical conformation. (D) Computer generated model for the structure of DNA containing a thymine dimer. (E) 5- Phenyl-indole-deoxyribose monophosphate paired opposite a thymine dimer in which the non-natural nucleobase is placed in an intrahelical conformation.

A

O- -O O- O- OP O- P O N O- O P O -O P O O- O O O P O N O P O O O OH O OH 5’ KD 5’

3’ 5’ 3’ 5’

kpol

O O O -O OP P O OP OH OH O- O- O- O O k 5’ chem 5’ N N

3’ 5’ 3’ 5’

B C

DE

83 The difference in kinetic parameters is significant as it indicates that gp43 does

not replicate the thymine dimer as either an abasic site or a thymine. Instead, the data

argue that the thymine dimer is replicated as a "hybrid" containing features common to

both the non-templating lesion and thymine. To explain such a phenomenon, we use the

models illustrated in Figure 3.8(A) to propose a unified mechanism for replicating

unmodified or damaged DNA. In the case of normal DNA replication, we propose that

the unmodified templating nucleobase is oriented in an extrahelical position that creates a

transient "void" mimicking an abasic site . This hypothesis is based upon kinetic evidence

using our non-natural nucleotides and structural models of various DNA polymerases

bound to nucleic acid (83-85). Our kinetic data indicate that large, bulky non-natural

nucleotides can easily fill the "void" produced by this transient intermediate. In fact, the low binding constants measured for their insertion opposite a true abasic site provides

further evidence for this mechanism. As seen in Figures 3.8(B) and 3.8(C), the non-

natural nucleobase of 5-PhITP can fill the void at an abasic site and is stabilized by π−π

electron interactions with the penultimate base pair.

Unlike binding affinity, however, kpol values are highly dependent upon the

presence of a templating nucleobase. When a normal templating base is present, the kpol values are slow since the shear bulk of large, non-natural nucleotides hinders the facile re-positioning of the templating nucleobase from an extra- into an intra-helical conformation. In contrast, the kpol values at an abasic site are significantly faster since the

lack of a templating nucleobase circumvents the need for re-positioning. Furthermore, the rate of the conformational change step is dependent upon the presence of π-electron

84 density (19) and is consistent with the favorable stacking interactions of the non-natural

nucleobase within the void of the abasic site [Figure 3.8(C)].

The dynamics of replicating of a thymine dimer are consistent with this model

especially if one considers that this lesion is a hybrid of a templating base and a non-

templating abasic site. We propose that the 3'-T of the lesion exists predominantly, but

not entirely, in an extrahelical position, while the 5'-T of the lesion still remains in an

intrahelical position. As shown in Figure 3.8(D), the extrahelical positioning of the 3'-T

creates an intermediate resembling an abasic site. However, the covalent bond between

the intrahelical 5'-T with the extrahelical 3'-T hinders the overall mobility of the lesion such that the rate constant for the pre-requisite conformational change step preceding phosphoryl transfer is significantly slower compared to that for a true abasic site.

In addition, these models indicate that the unique shape of the thymine dimer

influences ground-state binding. For example, the large bulky analog, 5-CH-ITP, is most

likely sterically hindered from binding in a proper orientation, and this is reflected in its

poor binding affinity (KD value >500 μM). In contrast, analogs containing large, flat

aromatic systems (5-PhITP and 5-NapITP) can interact more stably within the smaller

void caused by the 3'-T (Figure 3.8 (E)). Therefore, binding affinity increases as the

stacking interactions between the incoming nucleotide and DNA become optimal.

3.3.2. Rate-limiting step for replication of a thymine dimer.

To further compare and contrast the mechanism of translesion DNA synthesis, a

series of experiments were performed to evaluate the rate-limiting step during replication

opposite a thymine dimer. Previous studies using denaturing versus non-denaturing

85 quenching systems demonstrated that the conformational change preceding phosphoryl

transfer is rate-limiting for incorporation of non-natural nucleotides opposite an abasic site (35, 48). A similar approach was used here to monitor time courses in nucleotide incorporation using EDTA (non-denaturing quench) versus HCl (denaturing agent). Any observed differences in the amount and/or rate constants in product formation using these agents can provide information regarding the existence of various enzyme forms including E:DNA, E:DNA:dXTP, and E':DNA:dXTP that accumulate before the phosphoryl transfer step, E':DNAn+1:PPi.

Initial experiments were focused on the incorporation of 5-PhITP opposite the

thymine dimer. Time courses generated using KD concentrations of 5-PhITP (40 μM) are

provided in Figure 3.9. It is evident that the amplitude in product formation is

independent of quenching agent (210 nM using either HCl or EDTA). The identity in burst amplitudes indicates that phosphoryl transfer is not rate-limiting for the

incorporation of 5-PhITP opposite the lesion. However, the rate constant measured with

EDTA is 2.5-fold faster than that measured using HCl (compare 2.5 sec-1 versus 1 sec-1, respectively). This difference indicates that the conformational change preceding phosphoryl transfer is rate limiting for incorporation opposite the thymine dimer. A similar conclusion was made monitoring the incorporation of 5-PhITP opposite an abasic site (49). Thus, while the kinetic parameters for 5-PhITP incorporation opposite either lesion differ, the conformational change preceding phosphoryl transfer still remains the rate-limiting step regardless of lesion.

86 Figure 3.9 Time courses for the incorporation of 5-PhITP opposite a thymine dimer using EDTA or HCl as the quenching reagent. - Gp43 exo (1 μM) and 5'-labelled 13/20T=T-mer (250 nM) were pre-incubated, mixed with 10 mM Mg2+ and 30 μM 5-PhITP to initiate the reaction, and quenched with either 500 mM EDTA (g) or 1 M HCl (O) at variable times (0.05-5 seconds). After quenching with HCl, 100 μL phenol/chloroform/ iso-amyl alcohol was added to extract the polymerase and the pH of the aqueous phase was neutralized with the addition of 1M Tris/3M NaOH. Product formation was analyzed by denaturing gel electrophoresis followed by PhosphorImaging analysis.

250

200

150

100 14mer [nM] 50

0 0123456 Time (seconds)

To further evaluate this mechanism, I next monitored the incorporation of 5-CH-

ITP opposite a thymine dimer, an abasic site, and thymine using the different quenching

agents. As shown in Figure 3.10 (A), the amplitude in 5-CH-ITP incorporation opposite

the thymine dimer is independent of quenching agent. However, the rate constant

measured using EDTA is 2-fold faster than that using HCl (compare 0.06 sec-1 versus

0.03 sec-1, respectively). The difference in rate constants again indicates that the

conformational change preceding phosphoryl transfer is the rate-limiting step.

87 Figure 3.10 Time courses for the incorporation of 5-CH-ITP opposite a thymine dimer, an abasic site and thymine using EDTA or HCl as the quenching agent. Assays were performed as described in Figure 3.6 (A) EDTA(n) and HCl (O) quench opposite a thymine dimer (B) EDTA(n) and HCl (O) quench opposite an abasic site (C) EDTA (n)and HCl (O) quench opposite thymine.

A 500

400

300

200

100

0 0 20 40 60 80 100 120 140 160 Time (seconds)

B 500

400

300

200

100

0 0 102030405060 Time (seconds) C 500

400

300

200

100

0 0 20 40 60 80 100 120 140 Time (seconds)

88 To further investigate the rate limiting step, I performed identical additional experiments

monitoring 5-CH-ITP incorporation opposite the abasic site [Figure 3.10 (B)]. In this

case, the rate constants are essentially identical (2.6 sec-1 with EDTA versus 2.5 sec-1 with HCl) as are the burst amplitudes (400 nM versus 390 nM using EDTA or HCl, respectively). In marked contrast, the amplitude of product formation for 5-CH-ITP incorporation opposite T using HCl is reduced 40% compared to EDTA [Figure 3.10

(C)]. Furthermore, the rate constant using HCl is 3.5-fold slower than measured with

EDTA (compare 0.018 sec-1 versus 0.063 sec-1, respectively). The reduced rate constant

and 40% lower amplitude indicates that the conformational change and the phosphoryl

transfer step contribute equally toward limiting the overall rate constant for incorporation.

These data collectively indicate that the dynamics of replicating a thymine dimer

are more similar to an abasic site than the replication of a templating thymine. These

results have been interpreted with respect to the models provided in Figure 3.8. I propose

that the conformational change step represents structural reorganization of the incoming

nucleotide with the primer-template. During translesion DNA synthesis, it is easy to

envision that the non-natural nucleotide can fill the void at either an abasic site or a

thymine dimer and is properly oriented for phosphoryl transfer. This is not the case

during incorporation opposite a templating nucleobase as both the conformational change and phosphoryl transfer are rate-limiting for the insertion of bulky analogs such as 5-CH-

ITP. It is most likely that this bulky nucleobase hampers the re-positioning of the primer- template which also influences the rate of the phosphoryl transfer step.

89 3.3.3 Exonuclease proofreading activity at a thymine dimer.

Previously, our lab demonstrated that wild type gp43 rapidly removes dAMP

when placed opposite an abasic site at a rate that is ~30-fold faster than when dATP is

properly paired with thymine (81). This increase in proofreading ability was proposed to

reflect distortion of the primer/template caused by the lack of proper hydrogen-bonding and stacking interactions induced by the abasic site. Thus, a reasonable hypothesis is that

gp43 should excise mispairs at a thymine dimer with identical kinetics to those present at

an abasic site if both DNA lesions are functionally identical, i.e., both are non-

instructional. I evaluated this hypothesis by comparing the kinetics by which gp43exo+

excises natural and non-natural nucleotides paired opposite a thymine dimer, an abasic

site, or a templating thymine.

(a) Removal of natural nucleotides.

As such, first I performed reactions monitoring the enzymatic hydrolysis of dAMP

when placed opposite a thymine dimer or an abasic site employing single turnover

conditions in a rapid quench instrument as previously described (81). The time courses

for dAMP excision from both lesions are super-imposable and best defined as a single

exponential decay (Figure 3.11 ).

90 Figure 3.11 Enzymatic hydrolysis of dAMP paired opposite a thymine dimer and an abasic site by gp43 exo+ Experiments were performed by mixing a pre-incubated solution of 1 μM gp43 exo+:10 2+ mM Mg versus 250 nM 5’-labeled 14A/20T=T-mer ( ) or 14A/20SP-mer (□) DNA:10 mM Mg2+ (final concentrations) and terminating the reaction at various times by the addition of 350 mM EDTA. Each time course represents an average of two independent determinations. Time courses were fit to the equation for single exponential decay, y = Ae-kt + C, where A is the burst amplitude, k is the observed rate constant for product formation, and C is the end point of the reaction.

14-mer [nM]

Time (sec)

The rate constants for dAMP excision opposite an abasic site is 12.2 +/- 0.7 s-1 while opposite thymine dimer is 13.7 +/- 0.6 s-1. Although the measured rate constants are

identical, they are ~10-fold faster than the rate constant of ~0.74 +/- 0.07 s-1 for the

excision of dAMP paired opposite thymine. The faster rate constants likely reflect

distortion of primer-template junction caused by either lesion and argue that the thymine

dimer does resemble an abasic site. I propose that mispairs formed at either DNA lesion

enhance the enzyme's ability to partition the misaligned primer from the polymerase

active site into its exonuclease domain.

To provide further evidence for DNA partitioning, I attempted to measure the idle

turnover activity of the polymerase. Idle turnover is a process by which the polymerase

incorporates a dNTP and then excises the inserted dNMP in the absence of the next

91 required nucleotide triphosphate (86). Unfortunately, attempts to monitor the idle

turnover of dATP were futile since gp43 does not effectively incorporate this natural

nucleotide opposite the thymine dimer. As an alternative approach, I measured the rates

of dAMP excision from the penultimate base pair relative to the DNA lesion using

13/20T-mer, 13/20SP-mer, and 13/20T=T-mer as substrates. Time courses are provided in

Figure 3.12. A excision rate constant of 2.0 +/- 0.1 s-1 of dAMP excision at the

penultimate position with a template containing thymine dimer is ~2.7-fold faster than

0.74 +/- 0.09 s-1 of a template containing an abasic site or 0.82 +/- 0.07 s-1 for unmodified

thymine. This increased exonuclease activity suggests that gp43 "senses" the bulky DNA

lesion and facilitates partitioning of the DNA from the polymerase site into the

exonuclease active site at a higher frequency.

Figure 3.12. Primer degradation of DNA containing a thymine dimer, an abasic site, or thymine by gp43 exo+ All experiments were performed by mixing a pre-incubated solution of 1 μM gp43 + 2+ exo :10 mM Mg versus 250 nM 5’-labeled 13/20T=T-mer ( ) or 13/20SP-mer (x) or 2+ 13/20T-mer(□) DNA:10 mM Mg (final concentrations) and terminating the reaction at various times by the addition of 350 mM EDTA. Each time course represents an average of two independent determinations. Time courses were fit to the equation for single exponential decay, y = Ae-kt + C, where A is the burst amplitude, k is the observed rate constant for product formation, and C is the end point of the reaction

14-mer [nM]

Time (sec)

92 (b) Removal of non-natural nucleotides.

To investigate the proofreading capability of gp43 with 5-PhITP opposite the

thymine dimer, I performed the idle turnover experiments. This activity was quantified as

previously described (81) using a modified gel electrophoresis protocol monitoring the

amount of extension (13-mer to 14-mer) and subsequent excision (14-mer to 13-mer) of

the DNA as a function of time. Figure 3.13 provides representation time courses

comparing idle turnover of 5-PhITP opposite either DNA lesion. At an abasic site, the

primer is rapidly elongated to form 14-mer. This burst is followed by a short steady-state

phase of product accumulation that defines the kinetics of idle turnover. During the

process of nucleotide incorporation and excision, the concentration of 5-PhITP decreases until it becomes lower than the KD value. At this point, the polymerase cannot continue to incorporate opposite the lesion, and this causes the complete degradation of the 14-mer.

Idle turnover of 5-PhITP at a thymine dimer is markedly different because the steady- state phase for 14-mer accumulation is significantly longer than that measured opposite an abasic site. The accumulation of 14-mer reflects the inability of gp43 to excise the non-natural nucleotide when it is paired opposite the thymine dimer. The difference in degradation is arguably caused by the ability of the non-natural nucleotide to stack into a dead-end complex at a thymine dimer but not at an abasic site. The models illustrated in

Figures 3.8(C) and 3.8(E) is again useful in providing insight into the dichotomy in exonuclease activity. At an abasic site [Figure 3.8 (C)], the 5-phenylindole moiety stacks very well in an intrahelical conformation and conforms satisfactorily to the overall shape and size of a natural Watson-Crick base pair (49) . Thus, the enzyme can excise the non-

93 natural nucleotide at a rate constant that is comparable to the excision of dAMP opposite

T (81).

Figure 3.13 Comparison of idle turnover kinetics for 5-PhITP insertion opposite an abasic site versus a thymine dimer. + 1 μM gp43 exo was added last to a solution containing 250 nM 5’-labeled 13/20SP-mer 2+ (O) or 13/20T=T-mer (n), 10 mM Mg and 40 μM 5-PhITP. Reactions were terminated by the addition of 500 mM EDTA at time intervals ranging from 50-600 seconds. Nucleotide incorporation and excision were analyzed by denaturing gel electrophoresis

% 14-mer

Time (sec)

The model for 5-phenylindole opposite the thymine dimer (Figure 3.8 (E)) also shows the stacking interactions of the non-natural nucleotide within the void created by the covalent adduct. However, the shape of the 5-phenylindole: thymine dimer does not accurately mimic the overall shape/size of a natural base pair. At face value, this would suggest that the polymerase should degrade the mispair very efficiently. Indeed, it is demonstrated earlier that gp43 could easily excise dAMP when paired opposite a thymine dimer due to the distortion of the primer template. However, the inability to excise 5-

PhIMP argues against a model invoking simple distortion of the primer-template. I hypothesize that the close proximity of the 5'-T of the lesion to the phenyl moiety of the

94 non-natural nucleotide induces aberrant stacking interactions that can inhibit the vigorous

proofreading capabilities of gp43.

3.1.4 Removal of natural and non-natural nucleotides via pyrophosphorolysis.

Pyrophosphorolysis, the reversal of DNA polymerization, is an important enzyme

activity that can remove nucleotides from non-extendable primers in the absence of

exonuclease proofreading activity (87) . Reverse transcriptases and other viral

polymerases use pyrophosphorolysis to remove various chain-terminating nucleotide

analogs from their genomic material (88). This activity is typically associated with the

development of drug resistance to nucleoside analogs such as AZT and ddI (89, 90). I

quantified the pyrophosphorolytic activity of gp43 to evaluate its potential role in excising natural and non-natural nucleotides paired opposite normal or damaged templating nucleobases. Assays monitoring the excision of dAMP were performed under pseudo-first order reaction conditions using 500 nM DNA, 50 nM gp43 exo-, and 20 mM

- pyrophosphate (PPi). As shown in [Figure 3.14(A)], gp43 exo efficiently removes dAMP

paired opposite T through pyrophosphorolysis and recapitulates data previously reported

by Capson, et al. (5). This datum infers that the normal primer-template is in a proper

conformation that allows gp43 to bind it in the polymerization active site. Although the

enzyme is poised for elongation, the absence of the next correct dNTP allows the enzyme

to use PPi as a substrate to reverse the polymerization reaction.

Pyrophosphorolysis activity is not observed when dATP is paired opposite either

an abasic site or a thymine dimer [Figure 3.14 (A)]. This result is intriguing since both

mispairs activate the exonuclease activity of gp43 (vide supra). The dichotomy in activity

likely reflects the inability of gp43 to properly bind these mispairs in the polymerization

95 active site that is required for pyrophosphorolysis. We argue that the enzyme partitions

these mispairs into the exonuclease active site of gp43, and would be consistent with the

enhanced exonuclease activity of gp43 when dAMP is paired opposite the 3'-T of the

thymine dimer (vide supra). Partitioning of the mispair into the exonuclease active site

would also explain the reluctance of the polymerase to extend beyond the mispair by

incorporating opposite the 5'-T of the thymine dimer.

With this in mind, it is fascinating that 5-phenylindole can be removed by

pyrophosphorolysis when placed opposite an abasic site but not when it is paired opposite

a thymine dimer. These data have again been interpreted with the models provided in

Figure 3.8(C) and 3.8(E). At an abasic site, the 5-phenylindole moiety stacks well

opposite the lesion and mimics the overall shape and size of a natural Watson-Crick base

pair (49).Thus, gp43 binds this mispair in the polymerase active site and is poised for

elongation. However, in the absence of the next correct dNTP, the polymerase catalyzes

pyrophosphorolysis when supplied with PPi. Note that the rate of 5-PhIMP removal opposite an abasic site is ~8-fold slower than that measured for the pyrophosphorolysis of dATP paired opposite T [Figure 3.14 (B)]. This difference could reflect enhanced stability of 5-PhIMP through base-stacking interactions or the presence of an altered conformation that is not optimal for pyrophosphorolysis. Regardless, it is clear that the

non-natural nucleotide is processed more effectively when paired opposite an abasic site

compared to a thymine dimer.

96 Figure 3.14. Pyrophosphorolysis activity on unmodified or damaged DNA templates of gp43 exo- (A) Time courses for the excision of dAMP paired opposite T (n), dAMP paired opposite an abasic site (□), dAMP paired opposite a thymine dimer (¯), 5-PhIMP paired opposite an abasic site (O), and 5-PhIMP paired opposite a thymine dimer (d). Each time course represents an average of two independent determinations. (B) Comparison of the rates of pyrophosphorolysis for nucleotide excision opposite unmodified or damaged DNA templates

A

600

500

400

300

200

100

0 0 50 100 150 200 250 300 350 400 Time (seconds)

B 8 7 6 5 4 3 2 1 0 A:T A:SP A:T=T 5-Ph:SP 5-Ph:T=T

97 3.4 CONCLUSIONS

UV radiation causes a variety of covalently modified DNA lesions, the most prevalent form of which is the cis, syn thymine dimer (91). The hydrogen bonding groups required for base pair recognition are not altered at a thymine dimer, and this predicts that the adduct should be replicated as a miscoding lesion. However, thymine dimers also induce deformations in the helical structure of DNA (36) that create a cavity resembling an abasic site, a noninstructional DNA lesion. In this chapter, I used a series of non- natural nucleotides to demonstrate that the high-fidelity bacteriophage T4 DNA polymerase does indeed replicate the bulky lesion like an abasic site. Analogues containing large -electron surface areas are incorporated opposite either lesion ~1000- fold more efficiently than those analogues devoid of -electron density. One striking example is that the catalytic efficiency for incorporation of 5-PhITP opposite a thymine dimer is ~6000-fold higher than that for the incorporation of dATP. Similar results were obtained with the bacteriophage T7 DNA polymerase using dPTP as the non-natural nucleotide (78). However, the selectivity for incorporation of dPTP versus dATP opposite the thymine dimer is minimal since the reported value of ~15 (78) is significantly lower that that of ~6000 measured here using 5-PhITP and the T4 DNA polymerase. Regardless, it appears that both high-fidelity DNA polymerases use similar mechanisms to replicate a thymine dimer.

There are, however, notable differences in the kinetic parameters for these non- natural nucleotides that provide evidence that gp43 does not replicate a thymine dimer identically as an abasic site. Figure 3.15 provides comparative structure-activity relationships for the incorporation of several non-natural nucleotides opposite a thymine

98 dimer versus an abasic site. During incorporation opposite an abasic site, it is clear that

each of these nucleotide analogues can stack within the void present at the lesion. We

argue that these stacking interactions account for the relatively equal KD values of these

analogues at an abasic site (47, 49, 80, 81). For insertion opposite an abasic site, binding

affinity is independent of -electron density. Therefore, the lower catalytic efficiency for

5-CHITP compared to those for 5-CEITP, 5-PhITP, and 5-NITP results from a low kpol value caused by the diminished -electron surface area of 5-CHITP. As before, we argue that -electron surface area plays the preeminent role in enhancing the rate of the

conformational change step preceding phosphoryl transfer. As implied within the model

provided in Figure 3.15, the -electron surface areas of 5-CEITP, 5-PhITP, and 5-NITP

are also important for stacking interactions with the penultimate base pair.

These theoretical models can also be used to explain the differences in kinetic behavior at the thymine dimer. The catalytic efficiency for incorporation opposite the

thymine dimer is similar to that for an abasic site as both are linked with the overall - electron surface area of the incoming nucleotide. However, the rate constants for the conformational change step at a thymine dimer are considerably slower compared to those for an abasic site. We interpret these differences to reflect the influence of steric hindrance imposed by the bulky lesion. In the case of 5-CHITP, it is clear that the shear bulk of the incoming nucleotide prevents optimal interactions with the bulky thymine dimer. In addition, the effect of steric hindrance diminishes as the shape of the 5- substituent group becomes

99 Figure 3.15 Proposed structure-activity relationships for the enzymatic incorporation of various non-natural nucleotides opposite thymine dimer versus an abasic site. All models were constructed using Spartan '04 and are designed to illustrate the influence of -electron surface area, shape, and size on the overall catalytic efficiency for incorporation opposite either DNA lesion.

more planar as the degree of -electron density increases (compare 5-CEITP vs 5-PhITP

opposite the thymine dimer). One exception is 5-NITP as it is poorly incorporated

opposite the thymine dimer. Although 5-NITP is relatively planar, the model provided in

Figure 3.15 indicates that it preferentially interacts with the 5'-end of the thymine dimer

as opposed to stacking within the void caused by the thymine dimer. This difference may

account for the poor incorporation of 5-NITP opposite the bulky lesion. Collectively, these analyses indicate that biophysical parameters influencing binding affinity and the

rate of the conformational change step are influenced differentially by the dynamic

features of the DNA lesion.

100 Despite differences in polymerization activity, we obtained evidence that each

lesion is processed similarly with respect to exonuclease proofreading and

pyrophosphorolysis. The kinetics for excision of dAMP from either DNA lesion are

identical and significantly faster than the excision of dAMP paired opposite an

unmodified thymine. This enhancement likely reflects the enzyme's ability to partition

the misaligned primer from the polymerase-active site into its exonuclease domain.

Similarities are also observed in the pyrophosphorolysis activity of gp43. The enzyme

does not process dATP when the natural nucleotide is paired opposite either an abasic site

or a thymine dimer. The lack of pyrophosphorolysis at these naturally occurring mispairs

suggests that the primer-template is partitioned away from the polymerization domain.

The ability to partition DNA away from the polymerization domain and into the

exonuclease active site could contribute to the lack of extension beyond either class of

mispair.

In this respect, both thymine dimers and abasic sites are considered to be strong

replication blocks that lead to arrests in DNA synthesis (92, 93). Their ability to inhibit

high-fidelity DNA polymerases is proposed to reflect steric constraints imposed by the

"tightness" of the active site of this family of high-fidelity DNA polymerases (94). The

data provided here are consistent with this mechanism as geometrical constraints imposed

by covalent linkage of two adjacent thymines prohibit the lesion from properly fitting

within the polymerase's active site. From a biological perspective, the inability of high-

fidelity polymerases to incorporate efficiently dNTPs opposite either DNA lesion is undoubtedly an important step in preventing translesion synthesis. However, we propose that the capacity of the DNA polymerase to partition mispairs away from its

101 polymerization domain has equally important ramifications on the biological outcome of translesion DNA replication. In prokaryotes and eukaryotes, DNA replication blocks induced by diverse DNA lesions can be rescued through the action of various error-prone

DNA polymerases (95, 96). Relevant examples include pol and Dpo4 which are error- prone polymerases possessing "loose" active sites (94) that can bypass thymine dimers

(94, 97). Paradoxically, the activity of error-prone polymerases at damaged DNA ultimately allows the cell to maintain genomic fidelity. However, to perform their biological tasks, these error-prone polymerases must first gain access to the lesion by displacing the high-fidelity DNA polymerase that initially encounters it during chromosomal replication. There are several models explaining how high- and low-fidelity

DNA polymerases can "switch" places with each other at a DNA lesion(98). Although these models differ with respect to the involvement of other proteins and/or post- translational modifications, it is clear that the high-fidelity polymerases must first stall at the DNA lesion and provides a signal to initiate these processes. We propose that signaling is achieved via the exonuclease and/or idle turnover activity of the DNA polymerase when it encounters a DNA lesion. Idle turnover, the futile cycle of nucleotide incorporation and excision, prevents the stable insertion of a dNMP opposite a template lesion and inhibits mispair elongation. Another important ramification is that the process may allow the DNA polymerase to remain "stalled" in the vicinity of the DNA lesion. In the bacteriophage T4 system, polymerase stalling may provide a direct link between replication and DNA recombination that is essential for the prevention of DNA damage

(99). Similar mechanisms may exist in eukaryotes with respect to physically coupling the proteins involved in recombination with those at a stalled replication fork (100). The

102 presence of error-prone DNA polymerases in eukaryotic systems likely provides an additional level of complexity toward the processing of damaged DNA. However, it is easy to envision that exonuclease proofreading and idle turnover may provide an essential link with these different classes of DNA polymerases. Evaluating the dynamics of this process will prove to be interesting to examine with regard to prevention of DNA damage through recombination and/or translesion DNA synthesis.

103

CHAPTER 4

SPECTROSCOPIC ANALYSIS OF TRANSLESION DNA SYNTHESIS

104 4.1 INTRODUCTION

Fidelity of DNA replication and its coordination with other cellular processes is

essential for the survival and proliferation of any living organisms. In this process, the

fidelity is achieved through correct nucleotide selection and insertion, removal of

misinserted nucleotides by exonuclease activity, and the dissociation of enzyme from

primer/templates that are misaligned due to the mispairing (5). This multistep process

minimises errors committed during replication. During DNA replication this complex

exonuclease proofreading process ensures that the errors are committed only 1 in 108 catalytic turnovers (101). Although this complex process is well defined with respect to the excision of misincorporated nucleotides during the normal DNA replication, the exact molecular mechanisms that prevent error generation during translesion DNA synthesis is yet to be elucidated.

In this chapter I report the quantitative evaluation the dynamics of exonuclease proofreading during translesion DNA replication using the bacteriophage T4 DNA polymerase as model system and employing the abasic site and thymine dimer as the pro- typical lesions. Specifically, 5-NapITP (Figure 4.1), a highly conjugated non-natural nucleotide is used as the reporter to decipher the dynamics of translesion DNA synthesis.

Figure 4.1 Chemical structure of 5-NapITP

O O O N -O P O P O P O O O- O- O- OH

105 Although considered to be a simple system, the T4 system shares functional

homology with pol δ (102) present in higher eukaryotic organisms and provides a useful

paradigm toward deciphering how the DNA polymerase process different lesions during

translesion DNA synthesis. During DNA replication, gp43 catalyzes the incorporation of

nucleotides in the 5'Æ3' direction and maintains high fidelity through the assistance of its

intrinsic 3'Æ5' exonuclease activity (103). Through this coupled process, the errors

generated are instantly excised to maintain the integrity of the genomic information.

During this exonuclease proofreading process, the primer is translocated from

polymerase site to the exonuclease site to remove the mispair by exonuclease activity.

Once the nucleotide is excised, the primer is returned to the polymerase active site to

continue the replication process (104). This transfer may occur by an intermolecular

reaction, without dissociation of the polymerase from the DNA, as has been observed for

the T4 DNA polymerase (105). Recent structural data of RB69, a bacteriophage DNA

polymerase, that shares 80% sequence homology to gp43, provides evidence for

substantial movements of the DNA within the enzyme since the polymerization and

exonuclease active sites are at least 40Å apart (106). This structural information

augments the known kinetic mechanism of the proofreading pathway of gp43 as

illustrated in Figure 4.2.

Figure 4.2 Kinetic scheme representing the exonuclease proofreading pathway. Step 1 represents the translocation of primer from polymersase site to exonuclease site Step 2 represents the excision of dNMP from the primer at the exonuclease site Step 3 represents the release of excised dNMP

pol pre exo exo E :DNAn+1 E :DNAn+1 E :DNAn: dNMP E :DNAn + dNMP Step 1 Step 2 Step 3

106 Even though this pathway involves several distinct steps, it is the translocation of

the primer terminus from the polymerase to the exonuclease active site (step 1) that is rate-limiting for excision of the nucleotide. However, it is still unknown if this step is

controlled by inappropriate shape complementarity, reduced base-stacking capabilities, or

a combination of these biophysical parameters of the incorporated nucleotides at DNA

lesions that activate the exonuclease proofreading activity.

In this chapter, I investigate the proofreading mechanism of gp43 polymerase

upon encountering an abasic site and thymine dimer. Specifically, I exploited the

changes in the fluorescence signals of 5-NapITP to identify the intermediary steps that

account the exonuclease proofreading activity adopted at an abasic site or thymine dimer

lesion.

107 4.2 MATERIALS AND METHODS

4.2.1 Protein purification, preparation of DNA substrates and synthesis of 5-NapITP

Wild-type gp43 and the exonuclease-deficient mutant gp43 (D219A) mutant were

purified and quanitified as described previously (55). The oligonucleotide containing a

cis, syn thymine dimer was synthesized by Trilink Biotechnologies (San Diego,CA). All other oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an abasic site, were synthesized by Operon Technologies (Alameda, CA). Single-stranded and double-stranded duplex DNA used in this study (Table 4.1) were purified and quantified as described previously. The non-natural nucleotide, 5-NapITP used in this study was synthesized and purified as described previously(80). The excitation (255 nm) and emission (349 nm) wavelength for 5-NapITP was determined using Fluorimeter instrument.

Table 4.1 Duplex DNA substrates used in this study

13/20T=T-mer 5’-TCGCAGCCGGTCC-3’

3’-AGCGTCGGCCAGGT=TCCAAA-5’ T=T denotes thymine dimer

13/20SP-mer 5’-TCGCAGCCGGTCC-3’

3’-AGCGTCGGCCAGGSPCCCAAA-5’ SP denotes an abasic site

13/20T- mer 5’-TCGCAGCCGGTCC-3’ 3’-AGCGTCGGCCAGGTXCCAAA-5’ X denotes C or T or G or A

108 4.2.2 Determination of rate constant for incorporation of 5-NapITP at an abasic site, thymine dimer and thymine by gp43 exo- using stopped-flow technique.

Time course monitoring the insertion of 5-NapITP opposite an abasic site, thymine dimer and thymine were obtained by using a stopped flow by the data collection software Stop Flow version 7.50 β (KinTek Corporation, Clarence, PA). The instrument is equipped with PMT (photomultiplier tube) and a 340 nm long pass filter to detect fluorescence signals >340 nm. The sample syringes were maintained at 25 oC by a

circulating water bath. Syringe A contained 1000 nM gp43 DNA polymerase, with fixed

concentration of 5-NapITP (4 μM), 10 mM Mg(OAc)2, 50 mM Tris-HCl (pH 7.4), and 5

mM DTT. Syringe B contained 500nM DNA in assay buffer containing EDTA (100 mM) and Mg(OAc)2 (10 mM). The syringe contents were rapidly mixed in the sample

cell and reaction was monitored by the changes in the fluorescence signals (excitation

255 nm emission 349 nm). The time courses shown are a result of averaging at least six

traces and each course was normalized to percent fluorescence intensity. Data obtained

were fitted to Equation 4.1.

y = Ae-kt + C (4.1)

Here A is the burst amplitude, k is the observed rate constant for initial product formation, t is time and C is the end point of the reaction.

4.2.3. Determination of rate constants for incorporation of 5-NapITP at an abasic site, thymine dimer and thymine using 32P assay

A rapid quench apparatus (KinTeK Corporation, Clarence, PA) was used to

monitor the time courses of 5-NapITP incorporation with DNA substrates provided in

109 Table 4.1. Experiments were performed under a condition in which the concentrations of

polymerase and nucleotide substrate were maintained in molar excess versus that of DNA

substrate. A typical reaction was performed by rapidly mixing an aliquot of a

preincubated solution of DNA polymerase (1000 nM) and 5-NapITP (4 μM) in assay

buffer containing 50mM Tris-HCl, EDTA (100 mM) with an equal volume of solution

containing DNA (500 nM) and Mg (OAc)2 (10 mM). After the solutions had been mixed

in the rapid–quench-flow instrument for time intervals ranging from 0.1-60s, the

reactions were quenched by the addition of EDTA (350 mM) through the third syringe of

the instrument. The reaction products were then analyzed, as described above. Data

obtained for single-turnover DNA polymerization assays were fitted to Equation 4.2

y = A (1-e-kt) + C (4.2)

Here A is the burst amplitude, k is the observed rate constant for initial product

formation, t is time, and C is the end point of the reaction.

4.2.4 Idle-turnover measurements

Single turnover conditions were employed to measure the rates of excision of 5-

NapIMP opposite an:abasic site or thymine dimer or thymine base pair. The reaction was

initiated by mixing wild type gp43 (1 μM) with 500 nM DNA and 4 μM 5-NapITP in assay buffer containing EDTA (100 μM) and 10 mM Mg(OAc)2. Five hundred

nanomolar of DNA (13/20T=T-mer or 13/20SP-mer or 13/20T) was first preincubated with

5-NapITP (4 μM) in the presence of 30 μM dCTP. Due to the nature of the 13/20-mer

DNA substrate (Figure 4.1), dCMP insertion opposite G at position 13 in the template

maintains a usable primer template for the insertion of the non-natural nucleotide

110 opposite the lesions (position 14). In all cases, the reaction was initiated by the addition

of 1000 nM gp43 exo+. Reactions were quenched with 200 mM EDTA at time frames

ranging from 5 to 600 seconds. The quenched samples were processed and the product

formation was analyzed as described (23).

4.2.5 Pre-steady state kinetic anlysis using a stopped flow apparatus.

A stopped flow apparatus was used to monitor the time course in 5-NapIMP

insertion using 13/20SP as the DNA substrate. A preincubated solution of 200 nM gp43

exo- polymerase and 2000 nM DNA (final concentrations) was mixed with an equal

volume of a solution containing 10 mM Mg(OAc)2 and 4 μM 5-NapITP (final

concentrations) in the same reaction buffer. The sample was excited at 255 nm, and the

fluorescence emission data were collected by using a cut-off filter that measures

fluorescence signals greater than 340 nm. The time courses shown are a result of

averaging at least six traces. The averaged time courses were fit to equation 4.3

y = Ae-kt +Bt +C (4.3) where A is the burst amplitude, k is the observed rate constant for initial product formation, B is the steady-state rate, t is time, and C is a defined constant.

4.2.6 Pre-steady state kinetic analysis using 32P assay.

A rapid quench apparatus (KinTeK Corporation, Clarence, PA) was used to

monitor the time courses of 5-NapITP incorporation with 13/20SP-mer DNA substrate

provided in Table 4.1. A preincubated solution of 100 nM gp43 exo- polymerase and

1000 nM DNA (final concentrations) was mixed with an equal volume of a solution

containing 10 mM Mg(OAc)2 and 4 μM 5-NapITP or 40 μM (final concentrations) in the

111 reaction buffer containing 50mM Tris-HCl, EDTA (100mM). After the solutions had

been mixed in the rapid–quench-flow appartus, the reactions were quenched with 350

mM EDTA through the third syringe at regular time intervals ranging from 0.01-60s. The

reaction products were then analyzed, as described (23). Data from this experiment were

then fit to equation 4.3 defining a burst in product formation followed by a steady-state

rate.

4.2.7 Measurement of exonuclease activity in the presence of next correct nucleotide

Time course for monitoring the insertion of 5-NapITP opposite an abasic site, and thymine were obtained by using a Stopped Flow apparatus by the data collection software

Stop Flow version 7.50 β (KinTek Corporation, Clarence, PA). The apparatus equipped with PMT (photomultiplier tube) and a 340 nm long pass filter to detect fluorescence emission >340 nm. The sample syringes were maintained at 25 oC by a circulating water

bath. Syringe A contained 1000 nM gp43 DNA polymerase, with fixed concentration of

5-NapITP (4 μM), 10 mM Mg (OAc)2, 50 mM Tris-HCl (pH 7.4), 5 mM DTT and

various concentrations of dCTP or dGTP . Syringe B contained 500nM DNA in assay

buffer containing EDTA (100 mM) and Mg(OAc)2 (10 mM). The syringe contents were

rapidly mixed in the sample cell and reaction was monitored by the changes in the

fluorescence (excitation 255 nm emission 349 nm). The time courses shown are a result

of averaging at least six traces and each course was normalized to percent fluorescence

intensity. Data obtained for single-turnover DNA polymerization assays were fitted to

Equation 4.1.

112 4.3. RESULTS AND DISCUSSION

5-NapITP, a non-natural nucleotide incorporated opposite an abasic site and thymine dimer with favorable kinetic parameters by gp43 DNA polymerase was used as the fluorescent probe to investigate the dynamics of exonuclease proofreading during translesion DNA synthesis. As illustrated in Figure 4.3, several macroscophic steps play a significant role in maintaining genomic integerity during translesion DNA synthesis.

These steps include the incorporation of 5-NapITP (Step A), enzyme dissociation (Step

B), exonuclease proofreading activity (Step C) and an enzyme translocation step (Step

D). Step A is a well defined scheme which includes 1) binding of non-natural nucleotide to the enzyme DNA complex 2) conformational change prior to phosphoryl transfer 3) phosphoryl transfer. Once these kinetic steps are achieved to incorporate the non-natural nucleotide, additional steps (Step B-C) play a significant role in reducing the error frequency to maintain the genomic integerity. Step C is the dominant step that has been attributed to proofreading through the associated exonuclease activity to excise the mispair. In addition to excising the misinserted dNMP, proofreading returns the polymerase to a correct primer/template junction, which allows the enzyme to renew

“correct” DNA synthesis. Another possibility is that the enzyme can dissociate from the

DNA ( Figure 4.3, Step B) due to the altered geometry of the formed mispair (107), the rate constant at which enzyme dissociates from the primer/template may be enhanced.

This activity will facilitate in recruiting the DNA repair proteins to remove the DNA lesions. If the mispair formed at DNA lesions satisfy the requirement of Watson-Crick base pair geomentry, the enzyme can translocate to the next templating position to continue with the DNA polymerization. (Figure 4.3, Step D)

113 Figure 4.3 Kinetic scheme illustrating the dynamics of incorporation and excision of non-natural nucleotide by gp43 exo+ at damaged DNA. Scheme A represents the 1) binding of dNTP to enzyme DNA complex 2) conformational change step prior to phosphoryl transfer 3) phosphoryl transfer Step B represents enzyme dissociation from the mispair Scheme C represents 7’) formation of pre-exonuclease complex 7’a) exicision of dNMP at exonuclease site 7’b) release of the dNMP Scheme D represents 7’’) enzyme translocation to the next templating position 7”a) binding of next correct nucleotide 7”b) polymerization of next templating nucleotide

Step A

123 E:DNAn E:DNAn:dXTP E’:DNAn:dXTP E’:DNAn+1:PPi

Step B 4 7 6 5 E + DNAn+1 E:DNAn+1 E’:DNAn+1 E’:DNAn+1 + PPi

7” 7’ Step D Step C

pre E’:DNAn+1:dXTP E :DNAn+1 7’’a 7’a E’:DNA :dXTP exo n+1 E :DNAn+1 7’’b 7’b E:DNA exo n+2 E :DNAn + dNMP

To examine these macroscopic steps with gp43 exo+ (exonuclease proficient), it is first necessary to investigate whether the dynamics of 5-NapITP incorporation is identical to gp43 exo- (exonuclease deficient). If both the enzyme forms yields similar kinetic parameters for the incorporation of 5-NapITP, then the kinetic differences observed with gp43 exo+ can be equated to the different macroscophic steps as illustrated in Figure 4.3

114 4.3.1. Enzymatic incorporation of 5-NapITP at an abasic site, thymine dimer and thymine

using gp43 exo-

Our analyses begin with comparing the dynamics of 5-NapITP incorporation

opposite an abasic site, thymine dimer and thymine using exonuclease deficient gp43

DNA polymerase. This analysis is important as it provides insights into whether exonuclease proficient gp43 DNA polymerase also behaves in a similar fashion as exonuclease deficient gp43 during the incorporation of 5-NapITP at an abasic site and thymine dimer. Time courses for 5-NapITP incorporation opposite an abasic site, thymine dimer and thymine were measured as described in materials and methods by using the stopped flow apparatus to measure the changes in the fluorescence signals as a function of time, while a rapid quench apparatus was used to measure the product

32 formation using P assay as described in materials and methods. The reported kquench as illustrated in Figure 4.4 are average of 5-10 consecutive analysis with identical reaction conditions using stopped flow analyses and the values were normalized to percent fluorescence intensity. Similarly, the product formation measured using 32P assay with a

rapid quench apparatus were normalized to percent product formation. The time courses

generated with stopped flow technique were then fit to equation 4.1 describing

exponential fluorescence decay, while the time course generated from 32P assay were fit

to equation 4.2 describing a hyperbolic curve. All of the kinetic parameters are

summarized in Table 4.2 and the data is illustrated in Figure 4.4.

115 Figure 4.4 Incorporation of 5-NapITP at an abasic site, thymine dimer and thymine by gp43 exo-.

Gp43 (1 μM) was preincubated with 5-NapITP (4 μM), 10mM Mg (OAc)2 and 500 nM DNA (13/20SP-mer (Blue) (A), 13/20T=T (Red) (B), and 13/20T (Green) (C)) was added to initiate the reaction. The reaction was excited at 255nm and emission at 349nm was monitored using 340 nm long pass filter in stopped flow apparatus. Similar conditions were employed the product formation using radio labeled assay and values are represented in black line.

A 120 120

100 100

80 80 % 14mer

60 60

40 40

20 20 % Fluorescence% Intensity 0 0 0 0.10.20.30.40.5 Time (sec)

B 120 120

100 100

80 80 % 14mer

60 60

40 40

20 20 % Fluorescence% Intensity 0 0 012345

Time (sec)

C 120 120

100 100

80 80 % 14mer

60 60

40 40

20 20 % Fluorescence% Intensity 0 0 0246810 Time (sec)

116 Table 4.2 Summary of kinetic rate constants of 5-NapITP incorporation at an abasic site, thymine dimer, and the thymine catalyzed by gp43 exo- DNA polymerase. ------Fluorescence quench 32P assay -1 -1 DNA kobs (s ) kobs (s ) ------

13/20SP-mer 5.45 +/- 0.02 5.6 +/- 1.2

13/20T=T-mer 0.981+/- 0.005 0.77 +/- 0.01

13/20T-mer 0. 234 +/- 0.001 0.25 +/- 0.02 ------

Figure 4.4A, compares the time courses for the incorporation opposite an abasic

site. The decrease in fluorescence as a function of time reflects base-stacking interactions

of the nucleotide associated with incorporation into duplex DNA. The time course

observed with stopped flow analyses is best defined as single exponential fluorescence

decay and yields an observed rate constant of 5.45 +/- 0.02 s-1. The time course in

product formation measured using 32P assay is also a single exponential process, yielding

a rate constant of 5.6 +/- 1.2 s-1. This identity in rate constants indicates that both assays are monitoring the same kinetic step, which is the conformational change step that occurs after nucleotide binding and prior to the phosphoryl transfer step. Similar data was obtained monitoring the incorporation opposite the thymine dimer (Figure 4.4B). In this

case, the rate constant of 0.981 +/- 0.005 s-1 for exponential fluorescence decay is 1.3 fold

faster than the rate constant of 0.77 +/- 0.01 s-1 obtained monitoring primer elongation by

the radiolabeled assay. The difference in rate constants suggest that flipping of 5-

NapITP (fluorescence quenching) from extrahelical to interhelical conformation occurs

prior to the phosphoryl transfer step (primer elongation). This result provides the first

117 evidence validating that the phosphoryl transfer step is at least partially rate-limiting for the incorporation of a nucleotide analog opposite a damaged nucleobase.

It is important to emphasize that significant differences were observed in the rate constants measuring 5-NapITP incorporation opposite an abasic site versus thymine dimer. Indeed, it is clear that the rate constant of 5.65 s-1 for incorporation opposite an abasic site is ~5 fold faster than that of 0.98 s-1 measured opposite the thymine dimer.

This difference agrees with our previous work indicating that the gp43 DNA polymerase does not use a identical mechanism to replicate a thymine dimer or an abasic site (68).

Indeed, this difference in the rate constants between an abasic site and thymine dimer prompted us to test the ability of the gp43 DNA polymerase to incorporate 5-NapITP opposite thymine, a natural templating nucleobase. The fluorescence quench rate constant of 0.234+/-0.001 s-1 is similar to 0.25 +/- 0.02 s-1 measured using 32P assay

(Figure 4.4C) (Table 4.2). This similarity in the rate constants also indicates that the conformational change step prior to phosphoryl transfer is rate limiting at an undamaged

DNA. In comparison, the rate constant of ~0.25 +/- 0.01 s-1 measured for fluorescence quenching of 5-NapITP opposite thymine is ~4 fold slower than at thymine dimer indicating that thymine dimer is also not replicated like a thymine. Collectively, the 4- fold difference in the rate constants of thymine dimer with an abasic site and 5-fold difference with thymine supports our earlier inferences indicating that the gp43 DNA polymerase processes thymine dimer like a “hybrid” between an abasic site and thymine

(68).

118 4.3.2 Enzymatic incorporation of 5-NapITP at an abasic site, thymine dimer and thymine using gp43 exo+

Exonuclease proofreading plays an important role in maintaining genomic fidelity as it can remove misincorporated nucleotides during DNA synthesis. The exact proofreading mechanisms, by which the misincorporated nucleotides are excised at various DNA lesions to maintain the genomic integrity, are currently unknown.

Therefore, we evaluated the ability of the gp43 exo+ to incorporate and then excise 5-

NapITP opposite an abasic site, thymine dimer and thymine and the results are compared with exonuclease deficient gp43 (gp43 exo-) in the previous section. Assays were performed with conditions as described in materials and methods 4.2.3. First, the stopped flow analyses were performed with gp43 exo+ opposite an abasic site to identify the differences in the fluorescence signals compared to gp43 exo-, and the generated time courses are illustrated in Figure 4.5. As illustrated in Figure 4.5, stopped flow analyses with gp43 exo+ exhibits an initial fluorescence quench phase that is similar to the fluorescence quench phase observed with gp43 exo-. In addition, the fluorescence quench phase, an additional intermediary lag phase and a fluorescence recovery phase are exhibited by gp43 exo+.

119 Figure 4.5 Time courses representing the incorporation of 5-NapITP opposite an abasic site by gp43 exo- and gp43 exo+ Gp43 (1 μM) was preincubated with 5-NapITP (4 μM), 10mM Mg (OAc)2 and 500 nM DNA 13/20SP-mer was added to initiate the reaction. The reaction was excited at 255nm and emission at 349 nm was monitored using 340 nm filter in stopped flow apparatus. Red line represents the exonuclease deficient enzyme catalyzed reaction while the blue line represents the wild type gp43 catalyzed reaction.

Lag Phase

Fluorescence Recovery Phase % Fluorescence Intensity

Time (sec)

Similar analyses were also performed at thymine dimer and thymine and the

corresponding fluorescence time courses at an abasic site (Blue), thymine dimer (Red),

and thymine (Green) are provided in Figure 4.6. The initial kquench is measured by fitting

the data to equation 4.1, while the rate constant for the fluorescence recovery (krecovery) were determined by substituting t1/2 (half life) of the recovery phase to the equation k=

0.693/t1/2. The corresponding kinetic parameters determined from the data are

summarized in Table 4.3.

120 Figure 4.6 Time courses representing the incorporation of 5-NapITP at an abasic site, thymine dimer and thymine by gp43 exo+. Gp43 (1 μM) was preincubated with 5-NapITP (4 μM), 10mM Mg (OAC)2 and 500 nM DNA (13/20SP-mer (Blue) (A), 13/20T=T (Red) (B), and 13/20T (Green) (C)) was added to initiate the reaction. The reaction was excited at 255nm and emission at 349nm was monitored using 340 nm long pass filter in stopped flow apparatus.

ity % Fluorescence Intens

Time (sec)

Table 4.3 Summary of kinetic rate constants of 5-NapITP incorporation at an abasic site, thymine dimer, and the thymine catalyzed by gp43 exo+ ------1 -1 DNA kquench (s ) krecovery (s ) ------13/20SP-mer 5.6 +/- 0.1 0.173 +/- 0.003 13/20T=T-mer 1.03 +/- 0.05 0.082 +/- 0.005 13/20T-mer 0.215 +/- 0.003 0.042 +/- 0.001 ------

121 The fluorescence time courses provided in Figure 4.6 illustrate the complexity of

nucleotide incorporation and excision of 5-NapITP opposite an abasic site, thymine dimer

and thymine by gp43 exo+. In all three cases, the first phase of the time course shows the decrease in fluorescence signals that reflects base-stacking interactions of the nucleotide as it is incorporated opposite DNA lesion. At an abasic site the rate constant for the first

phase measured with gp43 exo+ is 5.6 +/- 0.1 s-1 and is not significantly different from

5.45 +/- 0.02s-1 (Table 4.2) measured using gp43 exo-. Similarly, a rate constant of 1.03

+/- 0.01 s-1 measured at a thymine dimer is similar to 0.98 +/- 0.1 s-1 (Table 4.2), and a rate constant of 0.21 +/- 0.01 s-1 at thymine is similar to 0.234 +/- 0.005 s-1 (Table 4.2)

measured using gp43 exo- polymerase. Thus, the kinetics of nucleotide incorporation by gp43 DNA polymerase is identical in the absence or presence of exonuclease proofreading capabilities.

At an abasic site, the fluorescence recovery phase corresponds to a rate constant of

0.173 +/- 0.003 s-1, while for a thymine dimer and the thymine a rate constant of 0.082

+/- 0.02 s-1 and 0.042 +/- 0.01 s-1 (Table 4.3) were calculated respectively from their

+ respective t1/2 values. The fluorescence recovery phase exhibited by gp43 exo could

represent the excision of 5-NapIMP from the lesion through the exonuclease activity as

illustrated in Figure 4.3 Step C, since the liberated nucleoside monophosphate is highly

fluorescent. However, it is also possible that the fluorescence recovery phase could

represent the dissociation of gp43 exo+ from the duplex DNA after the incorporation of 5-

NapITP at the lesions (Figure 4.3 Step B), since active dissociation of gp43 from the

primer template containing 5-NapIMP will also exhibit the fluorescence.

122 4.3.3 Excision of 5-NapIMP at an abasic site, thymine dimer and thymine by gp43 exo+

To investigate whether the fluorescence recovery phase represent excision of 5-

NapIMP, idle turnover conditions were adapted to monitor the degradation of 14mer to

13mer with as described in materials and methods (81). Idle turnover is a process in

which the polymerase incorporates a dNTP and then excises the inserted dNMP in the

absence of the next required nucleotide triphosphate. This activity was quantified as

previously described using a modified gel electrophoresis protocol monitoring the

amount of extension (13-mer to 14-mer) and subsequent excision (14-mer to 13-mer) of

the DNA as a function of time (81). All experiments were performed using conditions in

which gp43 DNA exo+ is present in molar excess amount than the DNA substrate to

ensure that all DNA was bound with polymerase during the course of the reaction. Under these conditions, the rates of product formation reflect the kinetics of insertion and

excision rather than enzyme dissociation from the mispair. The idle turnover activity

provides a more accurate representation of in vivo conditions in which the polymerase should be bound and stalled at the sight of DNA damage (81). As illustrated in Figure

4.7, the attenuation in product formation at an abasic site (blue line) is significantly

slower than the time course monitoring the fluorescence recovery phase (black line).

Indeed, visual inspection reveals that the change in fluorescence is complete after ~10

seconds (black line) while enzymatic degradation of the 14-mer is not detected until after

~30 seconds (blue line). The dichotomy in fluorescence change and product formation is

consistent with a mechanism in which the incorporated 5-NapITP becomes “un-stacked”

in a pre-exonuclease complex prior to enzymatic hydrolysis in the exonuclease active

site. A similar phenomenon was observed at a thymine dimer, and thymine.

123 Corresponding excision data is illustrated in Figure 4.8. The kexo values were determined by fitting the values using KINSIM simulation software and summarized in Table 4.3.

Figure 4.7 Time courses representing the incorporation and excision of 5-NapITP opposite an abasic site. The black line reflects fluorescence quenching and recovery of 5-NapITP while the blue line reflects the amount of product formation measured via the 32P-radiolabel assay.

Excision % 14-mer % 14-mer

Fluorescence % Fluorescence Intensity recovery

Time (sec)

124 Figure 4.8 Time courses representing the incorporation and excision of 5-NapITP opposite an abasic site, thymine dimer and thymine using 32P assay. Gp43 (1 μM) was preincubated with 5-NapITP (4 μM), 10mM Mg(OAc)2, 30 μM dCTP and 500 nM DNA 13/20SP-mer (Blue) or 13/20T=T-mer (Red) or 13/20T-mer (Green) was added to initiate the reaction. The reactions were quenched with 200 mM of EDTA at various time intervals as processed as previously described in materials and methods.

% 14-mer

Time (sec)

Table 4.3. Summary of excision rate constants of 5-NapIMP at an abasic site, thymine dimer and thymine catalyzed by gp43 exo+ ------1 DNA kexo (s ) KinFitSim Value ------

13/20SP-mer (abasic site ) 0.0239 +/- 0.004

13/20T=T-mer (thymine dimer) 0.0122 +/- 0.004

13/20T-mer (thymine) 0.0026 +/- 0.0003 ------

125 From the Table 4.3, it is evident that the 5-NapIMP excision at an abasic site

(13/20SP-mer) is ~2-fold faster than at thymine dimer (13/20T=T-mer) and 9-fold faster

than at thymine (13/20T-mer). In comparison, these values are significantly different

from the fluorescence recovery rate constants measured with the stopped flow assay. At

-1 an abasic site, a kexo of 0.0239 +/- 0.004 s (Step C 7’b, Figure 4.3) is ~7 fold slower than

0.173 +/- 0.002 s-1 (Step C 7’, Figure 4.3) (Table 4.3) fluorescence recovery phase.

-1 -1 Similarly a kexo of 0.0122 +/- 0.004 s is ~7 fold slower than 0.082 +/- 0.003 s (Table

-1 4.3) measured at thymine dimer, while a kexo of 0.0026 +/- 0.0003 s measured at

thymine is ~16 fold slower than 0.042 +/- 0.005 s-1 (Table 4.3). Collectively, these

differences in the rate constants between fluorescence recovery and excision values

indicate the formation of pre-exonuclease complex prior to the excision process.

Furthermore the difference in the fluorescence recovery phases between the lesions also

suggests that the pre-exonuclease formation is dependent upon the stacking interactions

of 5-NapIMP with the lesions. The vast differences in the fluorescence recovery and

subsequent excision between different lesions again illustrates that the dynamics adapted

by gp43 polymerase is lesion dependent (68). The dichotomy in fluorescence change and

product formation is consistent with a mechanism in which the incorporated 5-NapITP

becomes “un-stacked” in a pre-exonuclease complex (Figure, 4.3, Step C, 7’) prior to

enzymatic hydrolysis in the exonuclease active site (104, 108-111).

4.3.4 Pre steady state analysis of 5-NapITP incorporation at an abasic site by gp43exo-

The idle turnover experiments measuring exonuclease activity indicated that the fluorescence recovery phase does not arise from the excision of 5-NapITP from the lesions. I next evaluated whether the recovery phase represent the active enzyme

126 dissociation (Figure 4.2, Step B) from the duplex DNA containing 5-NapIMP. First,

experiments were conducted using stopped-flow apparatus as described in materials and

methods with limiting amount of gp43exo- (200nM) preincubated with molar excess of

13/20SP-mer DNA substrate (2000 nM). A fixed concentration 5-NapITP (4 μM) was

rapidly mixed to initiate the reaction. As illustrated in Figure 4.9, the time course for 5-

NapITP incorporation opposite an abasic site is biphasic as characterized by a rapid

initial burst in 14-mer formation followed by a second, slower phase in primer elongation. This type of biphasic time course provides a clear indication of a two-step

reaction mechanism in which the rate-limiting step for enzyme turnover occurs after

phosphoryl transfer and reflects release of gp43 from the extended DNA. The data were

then fit to equation 4.3 to measure the rate constant (kobs) for the first round of catalysis

(burst phase), and the kcat (enzyme turnover) from the steady state phase. Fluorescence

burst amplitude of 0.627 +/- 0.033 determined from the pre-steady plot corresponds to

224 nM of gp43 exo-. This was determined from slope of a calibration curve (Figure

4.10) that were generated by an assay monitoring the amplitude difference with various

DNA concentrations (100-750 nM) with molar excess of gp43 exo- (1000 nM) in

presence of 4 μM 5-NapITP in stopped flow apparatus. The enzyme concentration

determined from the slope of the calibration curve were then used to determine the kcat value of 2.0 +/- 0.2 s-1 by dividing the steady-state rate by the enzyme concentration,

while the observed rate constant of the burst phase is 7.6 +/- 0.4 s-1. The stopped flow

assay with high concentration of 5-NapITP cannot be used to test the dependency of

nucleotide concentration on steady state phase, as this will give rise to inner filter effect.

In this regard, I performed pre-steady kinetic analysis using 32P assay in rapid quench

127 apparatus with 4 and 40 μM 5-NapITP nucleotide concentrations to determine the

dependency of nucleotide concentration on burst phase and kcat values. The data were fit

with equation 4.3 describing the burst phase followed by linear steady-state phase. The

-1 -1 data is illustrated in Figure 4.11. An initial burst amplitude of ~5.6 s +/-0.1 s and a kcat

of 0.77 +/- 0.3 s-1 were measured at a concentration of 4 μM of 5-NapITP. Even at

-1 saturating concentration of 5-NapITP (40 μM), a kcat of 1.0 +/-0.2 s was measured

which is similar to 0.77 +/- 0.3 s-1, suggesting that the enzyme turnover value is

-1 independent of the nucleotide concentration. The kcat of 1.0 s measured with a

radiolabeled assay and 2.0 s-1 measured using stopped flow technique is ~ 10-20 fold

faster than the 0.173 s-1 of fluorescence recovery phase (Table 4.3) observed opposite an

abasic site using gp43 exo+. These differences in the values suggest that the initial

fluorescence recovery as measured with gp43 exo+ does not arise from the enzyme

dissociation process (Figure 4.3, Step B). In this regard, our data indicate that the recovery phase represents the “un-stacking” of 5-NapIMP from the lesions and occurs through the Step C as illustrated in Figure 4.3.

128 Figure 4.9 Pre-steady state fluorescence quench time course in the incorporation of 5-NapITP opposite an abasic site. - Gp43 exo (200 nM) was preincubated with 10mM Mg(OAc)2, 2000 nM DNA 13/20SP- mer and 5-NapITP (4 μM), was added to initiate the reaction. The reaction was excited at 255nm and emission at 349nm was monitored using 340nM filter in stopped flow apparatus.

Burst Phase Intensity

Relative Fluorescence Steady-State Phase

Time (sec)

Figure 4.10 Calibration curve of amplitude versus DNA concentration. - Gp43 exo (1 μM) was preincubated with 5-NapITP (4 μM), 10mM Mg(OAC)2 and various concentrations of 13/20SP-mer (100-750 nM) was added to initiate the reaction. The reaction was excited at 255nm and emission at 349nm was monitored using 340 nm long pass filter in stopped flow apparatus. The differences in the amplitude based on the DNA concentrations used were plotted against the DNA concentrations to generate the linear calibration curve.

Amplitude Amplitude

13/20SP-mer [nM]

129 Figure 4.11 Pre-steady state time course representing the incorporation of 5- NapITP opposite an abasic site measured using 32P assay - Gp43 exo (100 nM) was preincubated with 10mM Mg(OAc)2, 1000 nM DNA 13/20SP- mer and 5-NapITP (4 μM (red) or 40 μM (blue)), was added to initiate the reaction. The reaction was quenched at regular time intervals (0-3 seconds) with 350mM EDTA and processed as previously described. 14mer [nM]

Time (sec)

4.3.5 Experiments with next correct nucleotide.

As illustrated in Figure 4.5, gp43 exo+ exhibited an intermediate lag phase in fluorescence signal prior to fluorescence recovery phase. As in case of an abasic site and thymine dimer, the lag phase is short lived with a life time of ~ 3 sec, while for thymine this lag phase is relatively long lived with a time frame of ~ 7 sec. First, this lag phase could represent the ability of enzyme to translocate to the next templating information after the successful incorporation of non-natural nucleotide, as 5-NapIMP at abasic site mimics overall shape and size of Watson-Crick base pair (Figure 4.3, Step D).

Experiments were performed to test this hypothesis by adding next correct nucleotide in addition to the 5-NapITP. Through this approach, we expected further

extension of lag phase with the addition of the next correct nucleotide. In case of an

abasic site, based on the nature of the template used (13/20SP-C), a fixed amount of 150

130 μM (KD value) of dGTP (next correct nucleotide) was added along with the 5-NapITP (4

μM) to monitor any significant changes in the lag phase. It should be noted that only a fixed amount of dGTP was used in this experiment to eliminate the complications that may arise in the fluorescence amplitudes. As illustrated in Figure 4.12, addition of dGTP did not significantly alter the lag phase. In fact, a fluorescence recovery rate constant of

0.198 s-1 measured in presence of next correct nucleotide is similar to 0.173 s-1 (Table

4.3) measured in absence of next correct nucleotide.

Figure 4.12 Time courses of 5-NapITP incorporation at an abasic site in presence of next correct nucleotide catalyzed by gp43 exo+ Gp43 (1μM) was preincubated with 10mM Mg(OAc)2 and 5-NapITP (4 μM) and the reaction was initiated with 500nM 13/20SP-mer. The reaction was excited at 255nm and emission at 349 nm was monitored using 340 nm long pass filter in stopped flow apparatus. Red line represents the reactions condition without dGTP, while green line represents reaction conditions that include 150 μM dGTP.

% Fluorescence Intensity

Time (sec)

131 Similar experiments were also performed at an undamaged template consisting of thymine; based on the nature of template (13/20T-G-mer) variable concentrations of dCTP (0-200 μM) were added to the reaction mixture. As illustrated in Figure 4.13, the addition of next correct nucleotide extends the life time of intermediary lag phase from 7 seconds to ~35 seconds, without altering the initial incorporation phase. The extended lifetime of the lag phase suggests that primer predominantly resides in the polymerase active site on addition of next correct nucleotide. We argue that this difference occurs through the ability of the enzyme to translocate to the next templating information while processing an undamaged template (Figure 4.3, Step D). Upon translocation, the enzyme can bind to the next correct nucleotide to facilitate the insertion and this activity prevents the immediate “un-stacking” of the 5-NapIMP incorporated at the penultimate position.

This data indicate that translocation of the enzyme to the next templating position is facilitated through the favorable stacking interaction of the templating base with the incoming nucleotide and we propose this movement could play an important role in the enzyme translocation event (Figure 4.3, Step D)

132 Figure 4.13 Time courses of 5-NapITP incorporation at a thymine in presence of next correct nucleotide. + Gp43 exo (1μM) was preincubated with 10mM Mg(OAc)2 and 5-NapITP (4 μM) and the reaction was initiated with 500nM 13/20T-mer. The reaction was excited at 255nm and emission at 349 nm was monitored using 320 nm long pass filter in stopped flow apparatus. Red line represents the reactions condition without dCTP, while green line represents dCTP of 50 μM, blue (100μM), black (150 μM) and purple (200 μM)

% Fluorescence Intensity

Time (sec)

Collectively, the difference in the lag phases in presence of the next correct nucleotide with a template containing abasic site and thymine indicates that translocation is facilitated only at an undamaged template. Unfortunately, similar experiments could not be performed at thymine dimer template. In the case of thymine dimer, the addition of high concentrations dATP would compete with 5-NapITP incorporation at 3’thymine of thymine dimer and hence will complicate the analysis. Though our experiments indicate there are differences in the translocation phase based on the nature of the DNA lesions, further experimentation is required to fully define this intermediary lag phase.

133 4.3 CONCLUSIONS

Exonuclease proofreading mechanism of high fidelity DNA polymerase plays an

eminent role in maintaining genomic integrity (103, 110, 112). In this study, I

demonstrated that the microscopic events associated with exonuclease activity can be

monitored by the changes in fluorescence signal generated by the gp43 DNA polymerase

using 5-NapITP as the fluorescent probe at distinctly two different DNA lesions. The

data presented here indicates that the exonuclease mechanism is mediated through the

formation of a pre-exonuclease complex prior to enzymatic hydrolysis in the exonuclease

active site. As illustrated in Figure 4.5, the first step involves the incorporation of 5-

NapITP, and the second step is the formation of a pre-exonuclease complex prior to the

hydrolysis. The formation of a pre-exonuclease complex is detected by the recovery in 5-

NapIMP through the un-stacking process and its rate constants are completely driven by

the stacking interactions with the DNA lesions.

I have used these kinetic data to propose a model illustrated in Figure 4.14. It takes

into account many of the established features of DNA polymerization. In this mechanism

the first point for generating catalytic efficiency and fidelity of nucleotide incorporation

occurs during the binding of dNTP to the polymerase:DNA complex (Step 1). After

dNTP binding, a conformational change occurs prior to phosphoryl transfer (Step 2). The

molecular events underlying this mechanism have been interpreted with the formation of

hydrogen bonds between base pairs. In this model, the incoming dNTP directly pairs

opposite its complementary template partner so that the enzyme catalyzes the transesterification reaction only if the alignments between the two base pairs are correct.

As such, misincorporation events are predicted to rarely occur since the inability of the

134 functional groups to line up properly either perturbs ground state binding (step 1) and/or

reduces the

Figure 4.14 Proposed model for the exonuclease proofreading activity of gp43 DNA polymerase at DNA lesions.

Step 1 Step 2 Step 3 Step 4

Epre:DNAn+1 Eexo:DNA+dXMP E:DNA+dXTP E’:DNA+dXTP E:DNAn+1

Step 1 Conformational change step prior to phosphoryl transfer Step 2 Phosphoryl transfer step Step 3 Formation of pre-exonuclease complex prior to excision Step 4 Excision of dXMP

rate of the conformational change (step 2). This step is followed by a phosphoryl transfer

step to form the mispair (step 3). Since the 5-napthylindole: abasic site mispair mimics

the overall shape/size of a natural base pair, one would expect this behavior to allow the

polymerase to move to the next templating position to continue with the DNA

polymerization. However our analysis with next correct nucleotide suggests that enzyme

does not translocate to the next templating position after the successful incorporation

opposite an abasic site. We propose that this perturbation in return activates the

exonuclease activity to “un-stack” the 5-NapIMP (step 4) opposite an abasic site prior to

hydrolysis (step 5). Since, the un-stacking of 5-NapIMP is relatively easier from an abasic site than opposite a thymine dimer or thymine, the recovery phase occurs much faster, and leads to faster excision. However, opposite thymine, the aberrant stacking of

135 the 5-NapIMP and the translocation to the next templating position prevents the recovery

phase and thus slows the excision rate constants.

Our kinetic data suggests that translocation of DNA polymerase to the next

templating position is facilitated at an undamaged template. Based on our kinetic data,

we propose that flipping of the undamaged template information with the synchronized flipping of incoming nucleotide allow the enzyme to translocate to the next templating information. Since at thymine dimer, the 3’ T and 5 ‘T are covalently linked, the flipping

movement of the 3’T is highly hindered, and this severely hampers further movement of the enzyme. Similarly, the absence of templating information at an abasic site also hampers the traslocation of the enzyme to the next templating information. In this regard,

at lesions, the activation of exonuclease activity is mediated through the perturbation in

translocation to the next templating information. Since, translocation is hindered at a

thymine dimer and abasic site, the rate of nucleobase “un-stacking” and excisions are

correspondingly faster compared to thymine.

136

CHAPTER 5

CONCLUSIONS AND FUTURE DIRECTIONS

137 5.1 CONCLUSIONS

The main purpose of my thesis has been to evaluate the biophysical features that

control the DNA polymerases ability to perform translesion DNA synthesis. Biophysical

features such as hydrogen bonding, steric constraints, and base-stacking interactions are

proposed to control the DNA polymerases ability to incorporate nucleotides during the

bypassing of a lesion. Understanding the contributions of these individual biophysical

features is important both to the basic science of biological processes and to many useful

applications in biomedical research and biotechnology. From a basic science point of

view, these studies indicate which biophysical features govern a DNA polymerase is

ability to facilitate misincorporation at a specific lesion which may result in the

introduction of mutations. From a biomedical perspective, these studies provide a

foundation for the development of non-natural nucleotides as chemotherapeutic agents

capable of enhancing the effects of DNA-damaging agents by inhibiting pro-mutagenic

DNA replication. Undoubtedly, the present studies have added to our understanding of

translesion DNA synthesis. Specifically, I have demonstrated that base-stacking

contributions play a significant role in guiding DNA polymerases to perform translesion

DNA synthesis.

In chapter 2, using gp43 exo- (exonuclease deficient) enzyme I have evaluated which biophysical feature of the incoming nucleotide is important for its incorporation at an abasic site using a set of alkylated and un-natural purine nucleotides. In these studies,

I demonstrated that the nucleotides with greater base-stacking capabilities (extended π-

electron surface area and hydrophobicity) are incorporated opposite the lesion several

fold more efficiently compared to dATP. In this regard, the base-stacking features of the

138 incoming nucleotide enhance the rate of the enzyme-mediated conformational change

step (step 2 in figure 2.1) prior to the phosphoryl transfer step which enhances the

incorporation efficiency at an abasic site. Furthermore, I also demonstrated that

nucleotides with greater base-stacking capabilities are also extended 10-fold faster than

other analogs that are more hydrophilic and/or contain less π-electron density. These data demonstrate that incorporation and further elongation of a nucleotide at an abasic site by gp43 DNA polymerase is governed primarily through its associated π-electron density. Collectively, these data are consistent with the “base-stacking” model proposed by our lab (Figure 1.14).

In Chapter 3, I provided evidence that the thymine dimer is processed similarly to a non-templating abasic site. In both cases, the catalytic efficiency for the incorporation of a nucleotide is influenced by the π-electron surface area of the incoming nucleotide.

However, there are distinct differences in the kinetic parameters between an abasic site and thymine dimer, which indicate subtle differences in the mechanism for their misreplication. Thus, the binding affinity for non-natural nucleotides at a thymine dimer is linked to the overall π-electron surface area of the incoming non-natural nucleotide.

However, size and hydrophobicity mainly influence binding affinity at an abasic site.

The kpol step (step 2 in Figure 3.8 A) is also considerably slower opposite the thymine

dimer compared to an abasic site. Taken together, these observervations reflect the

influence of steric hindrance imposed by the bulky thymine dimer.

As described in Chapter 3, despite the differences in polymerization activity, each

lesion is processed similarly with respect to exonuclease and pyrophosphorolysis activity.

The kinetics of dAMP excision from either lesion is identical and significantly faster than

139 the excision of dAMP paired opposite an unmodified thymine. This enhancement likely

reflects the enzyme’s ability to partition the misaligned primer from the polymerase-

active site into its exonuclease domain. Removal of non-natural nucleotides also provides insight into the mechanism of this activity. 5-PhITP paired opposite an abasic site can be excised via exonuclease or pyrophosphorolysis. This phenomenon likely reflects the ability of this mispair to mimic the size and shape of a natural Watson-Crick pair (Figure 1.3). However, the enzyme does not excise 5-PhITP when paired opposite a thymine dimer. This likely reflects the helical distortion caused by the aberrant stacking

of 5-PhITP with 5’Thymine of the lesion.

In chapter 4, I extended my studies towards understanding the exonuclease

proofreading activity (Step C in Figure 4.3) adapted by gp43 DNA polymerase during

translesion DNA synthesis. The 3’Æ5’ exonuclease activity of high-fidelity DNA polymerases has been shown to modulate the processing of DNA lesions to remove the errors generated during the DNA replication (113) . Thus far, the complete dynamics of exonuclease proofreading at various DNA lesions are poorly understood. I used 5-

NapITP (Figure 4.1), the non-natural nucleotide incorporated opposite an abasic site and thymine dimer with favorable kinetic parameter as a fluorescent probe to detect the intermediary steps during exonuclease activity. I showed, for the first time the exonuclease proofreading activity of gp43 DNA polymerase at two different DNA lesions with an assay that continuously monitors the incorporation of 5-NapITP and the formation of a pre-exonuclease complex that leads to the excision of the 5-NapIMP.

These data also suggest that the formation of the “pre-exonuclease” (step C, 7’a in Figure

4.3) complex depends upon the stacking interactions of the 5-NapIMP at the lesions. The

140 data summarized in this chapter suggests that the thermodynamically favorable 5-

NapIMP:SP (Figure 3.5A) base pair is excised faster than the thermodynamically

unfavorable 5-NapIMP:T=T (Figure 3.5B) base pair indicating that shape

complementarity is not the primary feature that activates exonuclease proofreading. In addition, I obtained preliminary data to indicate that the translocation step (Figure 4.3,

Step D) of polymerase to the next templating position is hampered after the successful

incorporation of 5-NapITP at an abasic site and thymine dimer while it is favored at an

undamaged template.

5.2 FUTURE DIRECTIONS

My studies in Chapter 3 indicate that covalently linked DNA lesions such as cis,-

syn thymine dimer are not processed completely like an abasic site by gp43 DNA polymerase. This leads to an interesting hypothesis that all covalently linked lesions may be processed in a non-instructional manner by gp43 DNA polymerase. Accordingly, a lesion that might be expected to behave in a similar fashion as a cis,syn thymine dimer is the cis-platinum adduct of GG (Figure 5.1) that has its two bases covalently linked by platinum via the N7 position of G. Based on the kinetic evidences with non- natural nucleotides at cis,syn thymine dimer, I expect that a cis-plantinum adduct will also be replicated in a similar fashion with a similar preference for non-natural nucleotides by gp43 DNA polymerase. Based on the model proposed for thymine dimer, I predict that there will be differences in KD and kpol values because the adducted GG lesion is sterically larger than thymine dimer and would cause larger distortion in the active site residues.

141 Another lesion that would be of great interest is 7-bromomethylbenz[a]anthracene

N7 adducts of A and G (Figure 5.1). Previous reports by Dipple, et al. reveal that the T7

DNA polymerase preferentially insert adenine opposite both these adducts irrespective of

their size (114, 115). I predict that this lesion will also be processed in a manner similar

to an abasic site by gp43 DNA polymerase since the larger planar adducts may be flipped

out more freely from the DNA helical structure compared to a covalently linked lesion.

Analysis with our non-natural nucleotides will yield interesting results with the

differences in the nucleotide selectivity. For example, I hypothesize only the non-natural nucleotides with larger π-electron surface area will be preferred at cis platinated GG adducts while I predict that 5-NITP will be preferred more at a 7-bromo methyl- benz[a]anthracene adduct of A and G. A detailed kinetic analysis will provide the differences in KD and kpol values.

Figure 5.1 Chemical structures of DNA adducts (A) Cis, plantinum GG adduct (B) 7-bromomethylbenz[a]anthracene adenine adduct (C) 7-bromomethylbenz[a]anthracene guanine adduct

A B C

O O H2N NH2 NH HN H Pt H NH2 NH2 O H2N N N C C N N N Br N Br N NH N N dR Rd N N NH Rd Rd 2

A number of experiments remain for the completion of the studies described in

Chapter 4. To demonstrate that enzyme translocation (step D, Figure 4.3) to the next templating position is prevented at DNA lesions, it is critical to determine the burst rate

142 32 and kcat values using pre-steady state kinetics with P assay in the presence and in the absence of next correct nucleotide. I predict that the initial burst phase and steady state phase values will be unaltered at an abasic site and thymine dimer with and without the presence of next correct nucleotide, while I expect a plateau in steady state phase at thymine in the presence of the next correct nucleotide. This important experiment will

further support the hypothesis that the translocation to the next templating position by

gp43 DNA polymerase is prevented at an abasic site and thymine dimer, while it is

facilated at undamaged DNA.

Studies on exonuclease proofreading of gp43 DNA polymerase can be extended

further with other DNA lesions, such as cis plantinated GG adductus (Figure 5.1), 8- oxoguanine and other UV induced DNA lesions such as 6-4 photoproducts and Dewar products (Figure 5.2).

Figure 5.2 Chemical structures of (A) 8-oxoguanine, (6-4) photoproduct and (C) Dewar product

A B C O O O H CH 3 CH3 N HN HN NH OH O O N OH O N O N O N N N NH2 N Rd dR N H3C Rd H3C

These studies will provide a test of the hypothesis that the mode of exonuclease proofreading of gp43 DNA polymerase is universal for all other DNA lesions. The studies can also be extended to other DNA polymerases that possess intrinsic exonuclease

143 activity such as the Klenow fragment and mitochondrial DNA polymerase γ. One system that will be of great interest will be the Klenow fragment because it is proposed to obey a shape complementarity model at an abasic site (42). I predict that studies with the

Klenow fragment will provide interesting insights regarding the exonuclease proofreading dynamics between different DNA polymerases.

To begin to explore the base-stacking model with other DNA polymerases, we are collaborating with Dr. Mark Sutton, from the University of Buffalo, to characterize DinB

DNA polymerases on translesion DNA synthesis. DinB DNA polymerases are universally expressed in eukaryotes, archea and bacteria, and possess a loose active site to adopt a very open conformation to accommodate a variety of DNA lesions. This unique property of DinB provides an excellent model system to evaluate the dynamics of translesion DNA synthesis catalyzed by error-prone polymerases.

Preliminary experiments with DinB DNA polymerase from pseudomonas aeruginosa were performed to test its activity on an undamaged and damaged DNA. As illustrated in Figure 5.3, Pa-DinB preferably incorporates dCTP opposite G, and does not incorporate other natural nucleotides. This preliminary experiment demonstrates that Pa-

DinB does not misreplicate while bypassing an undamaged DNA template.

144 Figure 5.3 Incorporation of natural nucleotides at template G by Pa-DinB DNA polymerase Assays were performed using 100nM 13/20G-mer, 10 mM Mg(OAc)2 and 200nM of Pa- DinB and the reactions were initiated with100 μM dNTPs. The reaction was then quenched with 200 mM EDTA at 300 seconds and processed as previously described.

However, Pa-DinB is highly error-prone when processing a DNA containing an 8-

Oxoguanine lesion or an abasic site. As represented in Figure 5.4, Pa-DinB is able to

misincorporate natural nucleotides at 8-oxoguanine and an abasic site.

Figure 5.4 Incorporation of natural nucleoties opposite A) 8-oxoguanosine and B) an abasic site by Pa-BinB. Assays were performed using 100nM 13/20(8-OxodG)-mer or 13/20SP-mer, 10 mM Mg(OAc)2 and 200nM of Pa-DinB and the reactions were initiated with100 μM dNTPs at 8-Oxoguanine while 500 μM dNTPs were used at an abasic site. The reaction was then quenched with 200 mM EDTA at 300 seconds and processed as previously described.

A B

The representative gel figures suggests that the dATP and dCTP are equally preferred at

8-oxoguanine lesion, indicating that Pa-DinB is error-prone and tends to make mistakes

145 at a damaged template. The equal preference of dATP and dCTP at this lesion is

expected to occur through the syn and anti conformations of the 8-oxoguanine. A

detailed kinetic analysis has to be performed to demonstrate further differences in the

preference of the nucleotide. Also, I found that none of our non-natural nucleotides were

preferred at an 8-oxoguanine lesion, suggesting that Pa-DinB most likely relies on the hydrogen- bonding information of the lesion to incorporate a specific nucleotide. On the other hand, analyses with an abasic site suggest that dATP is most preferred by Pa-DinB and strictly obeys the “A-rule”. This observation is consistent with other DinB homologs such as E.coli Pol IV (116, 117). Among the various non-natural nucleotides analyzed, only 5-NITP is preferred at an abasic site by Pa-DinB (Figure 5.5).

Figure 5.5 Incorporation of non-natural nucleotides opposite an abasic site catalyzed by Pa-DinB. Assays were performed by preincubating 200nM Pa-DinB, 10 mM Mg(OAc)2 with 5’labeled 100nM 13/20SP-mer, 100 μM dXTP was added to initiate the reaction. After 300 sec, the reaction was quenched with 200 mM EDTA and processed as previously described.

This data indicate that Pa-DinB relies on base-stacking contributions to facilitate the

incorporation of the incoming nucleotide at an abasic site. However, the complete lack

146 of incorporation of other non-natural nucleotides that contain the extended π-electron system, such as 5-CEITP, 5-PhITP, and 5-NapITP, deviates from a base-stacking model.

Similarly, lack of IndTP and 5-FITP incorporation also suggests that the incorporation of

5-NITP is not primarily favored through hydropbhobic interactions. In this regard, it is possible that DinB relies on nucleotides with a smaller π-electron system. Additional experiments with other non-natural nucleotides with a smaller π-electron system such as

5-ethyleneITP and 5-cynITP need to be performed to test this hypothesis. A detailed kinetic analysis will provide further insights into the features that stimulate Pa-DinB

DNA polymerases to perform the translesion DNA synthesis.

DNA polymerases remain one of the most attractive drug targets in biological systems as they rely on biophysical features to facilitate nucleotide incorporation at DNA lesions to promote mutagenic events. Our studies indicate that gp43 DNA polymerase obeys base stacking model to facilitate nucleotide incorporation at an abasic site and thymine dimer. We can use our non-natural nucleotide library to screen other DNA polymerases such as the Klenow fragment, mitochondrial DNA polymerase γ, pol β and other error prone polymerases such as pol ηand pol κ to identify the biophysical features that they depend on for their activity during translesion DNA synthesis. We propose that we can exploit these individual biophysical features required by different DNA polymerases to misreplicate at DNA lesions by altering the base-stacking surface area, hydrophobicity and size of the non-natural nucleotides. The knowledge that will be gained from these studies will lead in the development of a potent and specific inhibitor for various DNA polymerases to prevent mutagenic events.

147 Current Chemical Biology, 2007, 1, 241-264 241

Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity

Babho Devadoss1 and Anthony J. Berdis*,2

Departments of 1Chemistry and 2Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA Abstract: DNA polymerases catalyze the addition of mononucleotides into a growing polymer using a DNA template as a guide for di- recting each incorporation event. The efficiency and fidelity of this biological process have been historically attributed to the ability of the DNA polymerase to coordinate proper hydrogen-bonding interactions between the incoming nucleotide with the templating nu- cleobase. However, the strength of this model has been weakened since several laboratories have demonstrated that non-natural nucleo- tides, i.e., those devoid of typical hydrogen-bonding capabilities, can be utilized by DNA polymerases with varying degrees of efficien- cies. This review provides a comprehensive summary of current research efforts leading to the development and implementation of these analogs as probes for DNA polymerase function and activity. The ability of various non-natural purines and pyrimidines to be incorpo- rated opposite templating nucleobases suggests that polymerization efficiency is not directly influenced by hydrogen-bonding interactions but rather by the overall shape and size of the formed base-pair. Conflicting evidence is obtained when the dynamics of nucleotide incor- poration is assessed using nucleic acid containing permutations in hydrogen bonding capabilities or completely devoid of these interac- tions. With respect to replication opposite an abasic site, it appears that the -electron surface area and desolvation properties of the in- coming nucleotide play a significant role for facilitating incorporation. This information has lead to the development of new models for DNA polymerization as well as toward strategies for novel biotechnology platforms and unique chemotherapeutic agents.

DNA replication is the process of duplicating DNA to make two Although the chemical mechanism of this reaction is well- identical copies so that one copy will be passed along to each daugh- defined, questions still exist as to how the polymerase maintains the ter cell after cell division. This complex biological process is cata- incredible degree of substrate "fidelity" during each incorporation lyzed minimally through the action of at least one DNA polymerase event. These enzymes are enigmatic as they maintain a remarkable that adds mononucleotides into a growing polymer (primer) using a degree of specificity despite the fact that the substrate requirement DNA template as a guide for directing each incorporation event (Fig. changes during each cycle of dNTP incorporation. Paradoxically, the 1). enzyme must maintain a high degree of selectivity to insert only one OH DNA polymerase OH of four potential dNTPs opposite a template place while it must also possess an extraordinary degree of flexibility to recognize four dis- ACGT + dNTP ACGTN + PPi TGCAYCTA TGCAYCTA tinct pairing partners. Furthermore, the polymerase performs the re- petitive cycle of base-pairing, phosphodiester bond formation, and Fig. (1). The process of DNA polymerization is typically a template-dependent translocation at a rate of nearly 1000 bp/sec [3]. Indeed, the combina- process. tion of accuracy and speed places the DNA polymerase in the hierar- DNA polymerases are arguably one of the most intricate catalysts chy of most efficient enzymes. found in nature as they represent a complex example of how enzymes This review will focus on three interrelated areas regarding the achieve rate enhancement with unique requirements in substrate rec- dynamics and mechanism of DNA polymerization. The first section ognition. As illustrated in Fig. 2, the chemistry of DNA polymeriza- will focus on understanding the molecular mechanism by which po- tion is a simple transesterification reaction [1] in which the - lymerases achieve the high degree of substrate specificity during the phosphate of the incoming 5'-deoxynucleoside triphosphate (dNTP) catalytic cycle and how defects in these activities can inadvertently undergoes nucleophilic attack by the 3'-OH of the primer strand of lead to disease development. The second aspect will explore efforts to the nucleic acid. The reaction is catalyzed with the participation of expand the repetoire of base pairing capabilities through permutations active site carboxylate residues that coordinate two metal ions. It is in hydrogen-bonding functional groups present on existing nucleo- proposed that one metal ion aids in the abstraction of the proton from tides. A major focus, however, will be on the use of nucleotides that the 3'-OH [2] while the other participates as a Lewis acid to coordi- are completely devoid of hydrogen-bonding potential. The third sec- nate the 
 phosphate groups of the incoming dNTP. tion will provide a short discussion of future directions that empha- size promising aspects in this field of research. This will focus on the

applications of the nucleobase analogs toward developing novel bio-

CH2 CH2 H C CH2 CH 2 CH2 C C -O 2 C C C C O- -O O O- -O O P O- O- -O O -O O -O O O O O 5' O O O- 5' 5' O 2+ P 2+ O 2+ O Mg O O O P O O Mg -O P O P O Mg O P O O O O O O- P O O O P O O- P O O 2+ O P O P OH O P Mg -O HO O- -O -O O- OH O O O- O 2+ O O O Mg O O O O A A A C C C A A T T G T G G T T T O O O O O O O O O O- O- - 3' O- 3' O- O 3' O- O- O- O- O OP O P O P O O P O O OP O P O O OP O O P O O O O OP O O O O O O O O O O Fig. (2). Proposed chemical mechanism for phosphoryl transfer catalyzed by DNA polymerases.

*Address correspondence to this author at the Department of Pharmacology, Case West- polymers that can be used for the storage and transfer of genetic in- ern Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Tel: (216)- formation in addition to the development of unique therapeutic agents 368-4723; Fax: (216) 368-3395; E-mail: [email protected] designed to combat disease.

1872-3136/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. 148 242 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

DNA replication is the concept of hydrogen bonding interactions that are used to stabilize nucleic acid and contribute to base identification during DNA polymerization. In this case, the mutual recognition of A by T and of G by C involves hydrogen bonding interactions between each partner. At the atomic level, the NH groups of the bases are good hydrogen bond donors (denoted as d) while the electron pairs on the oxygens of the base C=O groups and on the ring nitrogens are

hydrogen bond acceptors (denoted as a). This is best exemplified in Fig. 4 showing the hydrogen-bond acceptor and donor patterns for an A:T and G:C base pair. As indicated for these preferred tautomers, the hydrogen bonding capability of an A:T base pair uses comple-

mentarity of d*a*(-) to a*d*a while the G:C base pair uses com- plementarity of a*d*d to d*a*a. This base pairing pattern is com- monly referred to as Watson-Crick pairing. In planar base pairs, the hydrogen bonding pattern yields a geometry that gives a interglycosyl distance (C-1' to C-1') of 10.60 +/- 0.40 Å and an angle of 55 +/- 2o between the glycosylic bonds for both the A:T and G:C base pairs (Fig. 5). This isomorphous geometry indicates that all four base pair combinations - A:T, T:A, G:C, and C:G - can exist within the same regular framework of duplex DNA.

dd NH O CH3 N 2 a d N N HN

N N dR (-) a O dR

a d N O H2N d a N NH N dR N d N a dR Fig. (3). Structural model of duplex DNA. NH2 O THE CHEMISTRY AND BIOLOGY OF DNA. Before discussing the Fig. (4). Hydrogen bonding interactions between natural nucleobases. mechanism by which polymerases perform replication, it is first nec- While Watson-Crick base pairing is the predominant pattern used essary to discuss the chemical nature of the substrates utilized in the to stabilize DNA, several other forms of base-pairing interactions reaction - deoxyribonucleic acid (DNA) and deoxynucleoside have been identified. Two of the more commonly referenced forms triphosphates (dNTPs). DNA is a complex biopolymer that is the include Hoogsteen and wobble base pairs. Fig. 6 compares the struc- carrier of genetic information. The double-stranded, helical chain tural similarities and differences that exist amongst Watson-Crick, consists of nucleotides linked in a linear fashion that are complements Hoogsteen, and wobble base pairs. The Hoogsteen pair (Fig. 6B) is of each other on the opposing strand with respect to hydrogen bond- formed by one base rotating 180o relative to the other. This rotation ing and steric interactions (Fig. 3). Nucleotides are the building makes the Hoogsteen pair not isomorphous since the pair has an 80o blocks of DNA and are composed of three basic components that angle between the glycosylic bond and a separation of 8.6 Å of the include a nitrogen heterocylic base, a pentose sugar, and a phosphate. anomeric carbons. In contrast, a "wobble" base pair does not involve The major bases of DNA are monocyclic pyrimidines including rotation of a base but rather a sideways shift of one base relative to its thymine (T) and cytosine (C) and bicyclic purines that include ade- positions in a normal Watson-Crick pair (Fig. 5C). In the case of a nine (A) and guanine (G). At the core of nucleic acid structure and wobble base pair formed between G and T, the resulting loss of a

o 1.88A 1.78Ao N NH2 O CH3 N O H N o 2 1.82A 1.86Ao N N HN N NH N N N N 1.82Ao N DNA ooO DNA DNA O ~55 ooNH2 ~56 ~53 ~56 DNA 10.6 +/- 0.3 Ao 10.2+/- 0.3 Ao

Major Groove Major Groove

NH O CH N 2 3 N O H2N

N N HN N NH N N N N DNA O DNA N DNA NH O 2 DNA Minor Groove Minor Groove

Fig. (5). Bond angles and glycosidic bond distances between natural base pairs. 149 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 243 hydrogen bond leads to a reduced stability which may be partially grooves contain functional groups that can participate in hydrogen offset by improvements in base stacking interactions caused by the bonding interactions with proteins such as the DNA polymerase. displacement. Hoogsteen base pairs appear to play an important role CONTRIBUTION OF THE DNA POLYMERASE TOWARD RATE EN- in DNA synthesis catalyzed by so-called error-prone DNA polym- HANCEMENT AND FIDELITY. Hydrogen-bonding, electrostatic, steric, erases such as pol iota [4] while wobble base pairs can be formed and desolvation interactions play important roles in maintaining the during promutagenic DNA synthesis of damaged nucleobases [5,6]. defined double helical nature of DNA. However, their relative roles A N NH2 O CH3 in achieving the incredible degree of specificity for forming nascent base pairs during the polymerization reaction remain rather poorly N N HN defined. Attempts to evaluate these forces on the kinetics of nucleo- N N tide incorporation have been performed using a combination of struc- DNA O DNA tural and kinetic studies. We shall first summarize the results of vari- ous kinetic studies since these efforts preceded structural determina- tions aimed at deciphering the mechanism of DNA polymerization at B N the atomic level. NH2 N O CH3 In general, kinetic studies quantify the rate of nucleotide incorpo- HN ration by measuring the ability of the DNA polymerase to elongate N N N the primer strand as a function of time. The vast majority of these DNA O DNA studies evaluate polymerization fidelity by comparing the rates of mispair formation with that for forming the preferred Watson-Crick base pair. The most convenient method involves measuring elonga- tion of the primer strand of duplex DNA using denaturing polyacry- C O CH3 lamide electrophoresis (reviewed in [8,9]). In such an experiment, the N O HN reaction is monitored by the elongation of 32P-labeled duplex DNA or N through the incorporation of -32P-dNTP into DNA. In most cases, N NH O DNA experiments are performed using a fixed concentration of a single N DNA nucleotide so that the primer (DNA ) is elongated by one nucleobase NH2 n to yield DNAn+1. As outlined in Fig. 7, the polymerase extends the Fig. (6). Structures of base pairs existing in (A) Watson-Crick, (B) Hoogsteen, DNA in the presence of Mg2+ and dNTP (Step A) and the products and (C) wobble pairing arrangements. are visualized by separating the products of the reaction (DNAn+1) Although hydrogen bonding interactions are arguably the most from substrates (DNAn) by denaturing gel electrophoresis (Step B). common physical feature associated with the conformation and The gel is then quantified using either X-ray or PhosphorImaging dynamic stucture of DNA, other physical features such as 
 techniques and product formation is quantified by measuring the ratio 32 stacking interactions, desolvation, and geometrical constraints of P-labeled extended (DNAn+1) versus non-extended (DNAn) contribute extensively to the stability of nucleic acid (reviewed in primer (Step C). [7]). Solvation energy is defined as the amount of energy required to Measurements of polymerization efficiency and fidelity are typi- remove water from a molecule. Hydrophobicity, on the other hand, is cally performed using steady-state kinetic approaches to evaluate defined as the tendency of a molecule to repel water. Although these three primary kinetic parameters: V , K , and V /K [9,10]. V terms are generally used interchangeably, they have different bio- max m max m max is defined as the maximal rate of the reaction that is typically ob- physical consequences toward stabilizing nucleobase interactions served using saturating concentrations of the dNTP substrate. An- during polymerization. As evident from the structures of duplex other commonly used parameter is k which is defined as the V of DNA, the interior of the helix is devoid of water and hence must be cat max the reaction divided by the concentration of DNA polymerase used in considered a hydrophobic environment. This hydrophobicity is neces- the reaction. k is a rate constant that reflects the rate-limiting step sary in order for direct hydrogen-bonding interactions to occur be- cat for polymerase turnover. The Michaelis constant, K , is the concen- tween the functional groups of the nucleobases. During the process of m tration of substrate required to reach one-half the maximal reaction of polymerization, it is clear that both the templating and incoming nu- the polymerization reaction. This kinetic constant can, under certain cleobase must be desolvated to allow formation of the prerequisite conditions, also provide information regarding the binding affinity of hydrogen-bonding network within the hydrophobic core of the DNA 1 a dNTP for the polymerase . The last parameter, V /K , reflects the helix. max m apparent second-order rate constant for productive substrate binding. Equally important are the -stacking interactions among the aro- This value is often associated with the well-known “specificity- matic bases. In the DNA helix, all nucleobases are stacked in an off- constant” for an enzymatic reaction and provides a measure of the set geometry that is effectively controlled by 
 electron interac- catalytic efficiency of the enzyme [11]. As shown in Fig. 8, these tions through their aromatic nature. It is important to emphasize that kinetic parameters can be easily obtained by monitoring the rate of each of the aforementioned features (hydrogen-bonding potential, - product formation by varying the concentration of dNTP and using electrons, solvation energies) are interrelated such that one parameter fixed concentrations of polymerase and DNA substrate. Specifically, influences the dynamic feature of the other. For example, it is diffi- reactions are performed under pseudo-first order reaction conditions cult to alter a hydrogen bonding functional group without inadver- in which the concentration of DNA and dNTP are in molar excess tently influencing the solvation energy of the base or influencing its with respect to the concentration of DNA polymerase. degree of aromaticity and/or base stacking capabilities. A more powerful method to evaluate the mechanism and dynam- As illustrated in Fig. 3, the double helix of DNA resembles an ics of both correct and promutagenic DNA synthesis is the applica- intertwining spiral staircase in which the bases are stacked above one tion of rapid chemical quenching techniques [12,13]. Briefly this another. Each base is rotated ~36o around the helical axis relative to technique allows one to monitor the polymerization reaction on a the next base pair. In this arrangement, about 10 base pairs make a millisecond time scale. Under these conditions, the time course for complete turn of 360o. The twisting of the two strands to form the double helix create a minor groove and a major groove (Fig. 5). The 1 minor groove is approximately 12 Å across while the major groove is Although this parameter is loosely associated with binding affinity, it is often erroneously referred to as an equilibrium binding constant of the substrate for polymerase. The K for Å m roughly 22 in width. As indicated, both the major and minor a substrate can be considered equivalent to the kinetic dissociation constant (Kd) only under conditions in which chemistry (or a kinetic step preceding chemistry) is completely rate-limiting for product formation. 150 244 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

Fig. (7). Assay protocols to measure the kinetics of DNA polymerization.

Fig. (8). Determination of kinetic constants using Michealis-Menten plot analyses. correct dNTP incorporation (dATP opposite T, for example) for dNTP concentration. Using this technique, values corresponding to many DNA polymerases is biphasic in which there is rapid primer kpol, Kd, and kpol/Kd can be obtained experimentally. Note that under elongation followed by a second, slower phase. This biphasic time these conditions, the kpol value reflects the rate constant for the first course is indicative of a two-step reaction mechanism in which a round of DNA polymerization (the burst phase) and should not be kinetic step after phosphoryl transfer is the rate-limiting step for po- confused with Vmax or kcat values which define the rates and rate con- lymerase turnover [14]. The first phase of the time course most likely stants, respectively, measured during multiple rounds of polymerase represents a conformational change step needed to align to incoming turnover. This difference is best illustrated using the T4 DNA polym- dNTP with the templating nucleobase. The second, slower phase erase as a model [17]. In this system, the kpol value for the "burst represents the release of polymerase from elongated DNA and is the phase" is approximately 100 sec-1 while that rate constant for polym- -1 rate-limiting step for enzyme turnover [15,16]. erase turnover (kcat) is approximately 50-fold slower at ~2 sec [17]. Using rapid quench techniques, it is possible to monitor the rate The difference in rate constants provides a clear indication that a step constant of the initial fast phase in product formation as a function of after phosphoryl transfer such as product release is rate-limiting for enzyme turnover. 151 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 245

dNTP 2 Conformational DNA Binding E:DNA E:DNA :dNTP Change n n 3 1 dNTP Binding E’:DNAn:dNTP Processive 8 DNA Synthesis Phosphoryl E + DNAn Transfer 4

7 E’:DNAn+1:PPi Enzyme PPi 5 Dissociation 6 E:DNA E:DNA :PP Conformational n+1 n+1 i Change Pyrophosphate Release

aasddfssaa Fig. (9). Kinetic mechanism for DNA polymerases.

Similarly, the Kd value obtained under these conditions can be by the bacteriophage T4 DNA polymerase is disfavored ~370,000- measured by monitoring the dependency of nucleotide concentration fold compared to correct incorporation of dATP opposite T [26]. The of the rate constant for product formation. Although this value is binding affinity for dATP opposite C is 1,100 μM and is ~110-fold similar to the aforementioned Km value, it provides a more accurate higher than the binding affinity of 10 μM measured for dATP incor- description for ground state binding of the incoming dNTP. Finally, poration opposite T [17,26]. In addition, the rate constant for the con- -1 the kpol/Kd term is analogous to Vmax/Km as both reflect the apparent formational change preceding chemistry step is ~0.03 sec and repre- -1 second-order rate constant for productive substrate binding and pro- sents a 3,300-fold reduction compared to the kpol value of 100 sec vide measures of the catalytic efficiency of the enzyme. measured for the correct incorporation of dATP opposite T [17,26]. -1 -1 Data from steady-state and transient kinetic studies have been Thus, the catalytic efficiency for creating a mismatch is 27 M sec while that for correct DNA synthesis is 107 M-1sec-1. The differences collectively used to describe DNA polymerization via the multiplica- -6 tive mechanism outlined in Fig. 9. This is an ordered kinetic mecha- in catalytic efficiencies of 2.7*10 correspond to a predicted error nism in which the DNA polymerase binds DNA prior to the binding frequency of one mistake every 1,000,000 incorporation events. It of dNTP. In this mechanism, the first point for generating catalytic should be noted that this calculated error frequency is close to that efficiency and fidelity of nucleotide incorporation occurs during the measured in vivo with the exonuclease deficient T4 DNA polymerase binding of dNTP to the polymerase:DNA complex (Step 2). After [27]. dNTP binding, a conformational change (Step 3) in the enzyme is An additional level of error discrimination results from proof- proposed to align the incoming dNTP into a precise geometrical con- reading of any formed mismatches by the 3’-> 5’ exonuclease activity formation for phosphoryl transfer (Step 4). Results from several inde- of the polymerase. This activity removes misincorporated nucleotides pendent experiments including pulse-chase [17] and fluorescence to regenerate a correctly base-paired primer/template and can reduce quenching techniques [18] indicate that the rate constant of ~100 sec-1 the error frequency by an additional102-103-fold [28]. Although this measured with the T4 DNA polymerase corresponds to a conforma- activity is important in maintaining fidelity, it will not be extensively tional change step preceding phosphoryl transfer. This kinetic step is discussed in this review since most of research highlighted here util- the major contributor to polymerization fidelity as it constitutes a key ize DNA polymerases that are devoid of exonuclease proofreading. kinetic point for error discrimination. The involvement of the con- The reader is referred to the following articles for a more comprehen- formational change is consistent with the proposed "induced-fit" sive review of exonuclease activity [29,30]. mechanism that imposes discrimination against the misinsertion of a STRUCTURAL MODELS ACCOUNTING FOR POLYMERIZATION EFFI- dNMP since misaligned intermediates may disrupt or alter the ge- CIENCY AND FIDELITY. Kinetic characterization of DNA polymerases ometry of the polymerase’s active site to prevent chemistry [19]. It is have provided the basic framework for understanding the dynamics also possible that error discrimination can occur during the phos- of correct and incorrect DNA synthesis. However, it was not until phoryl step (Step 4) such that it becomes completely rate-limiting 1985 that the first detailed structure of a DNA polymerase was re- [20]. Indeed, it is proposed that phosphoryl transfer is completely or ported by Tom Steitz’s group [31]. Their studies provided an exqui- partially rate-limiting for nucleotide incorporation with pol
[21] and site picture describing the molecular architecture of the enzyme. the RNA-dependent RNA polymerase from poliovirus [22]. Since then, an explosion in the field of structural biology has gener- The molecular events underlying this kinetic mechanism have ated hundreds of structures for various DNA polymerases in the ab- historically been interpreted with the formation of hydrogen bonds sence and presence of DNA and nucleotide substrates. between base pairs (reviewed in [23-25]). In this model, the incoming Based upon sequence alignment data and crystallographic analy- dNTP is proposed to directly pair opposite its complementary tem- ses, DNA polymerases can be divided into at least five distinct fami- plate partner so that the enzyme catalyzes the transesterification reac- lies denoted as A, B, X, Y, and reverse transcriptase (reviewed in tion only if the alignment between the two is correct. As such, misin- [32,33]). From a structural perspective, the most extensively studied corporation events are predicted to rarely occur since the inability of polymerase family is the A family which includes the Klenow frag- the functional groups to line up properly either perturbs ground state ments of DNA polymerase I from Escherichia coli [34-36] and Bacil- binding (Step 2) and/or reduces the rate of the conformational change lus subtillus [37-39], Thermus aquaticus DNA polymerase [40,41], (Step 3). At face value, the kinetic constants are consistent with this and the bacteriophage T7 DNA polymerase [42-44]. These structures model. For example, misincorporation of dATP opposite C catalyzed 152 246 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis indicate that all DNA polymerases share a common overall architec- THE USE OF MODIFIED NUCLEOTIDES AND NON-NATURAL NU- ture resembling a "right hand" containing fingers, palm, and thumb CLEOTIDES TO STUDY THE MECHANISM OF FIDELITY. Mutagenesis is subdomains (Fig. 10). The palm domain is responsible for catalysis of the process of changing the DNA base sequence of an organism's the phosphoryl transfer reaction and as such, is the most closely con- genome at a specific site. This is a complex process that occurs served structural feature amongst all polymerases. The palm domain minimally through the ability of the DNA polymerase to inappropri- contains at least two carboxylates that function to coordinate two ately insert a non-complementarity nucleotide opposite a templating catalytically essential metal ions that participate in phosphoryl trans- base, the ability of the formed mispair to be elongated, and the inabil- fer. The fingers domain interacts with the incoming dNTP as well as ity of various DNA repair enzymes to correct the mismatch prior to the templating base and thus plays an important role in maintaining the next round of DNA replication. As mentioned earlier, mutagene- fidelity. The thumb domain plays dual roles by positioning duplex sis can occur by the misincorporation of dATP opposite C that can DNA for the incoming dNTP as well as in the processivity and trans- ultimately lead to an A:T to G:C transition mutation. However, this location of the polymerase. Although the functions of the fingers and type of "simple" mutation is infrequent as high fidelity DNA poly- thumb subdomains are relatively conserved amongst DNA polym- merases misincorporate opposite un-damaged DNA roughly once out erases, they are widely divergent with respect to structural character- of every million incorporation events [46]. This extraordinary degree istics. These structural differences are proposed to account for nu- of fidelity has been proposed to reflect the influence of hydrogen- ances in the dynamics of nucleotide incorporation and intrinsic fidel- bonding interactions. At the atomic level, the ability of base pairs to ity of various polymerases [45]. properly form is governed by the various functional groups (keto oxygen groups, heterocyclic nitrogens, and exocyclic amine groups) that are present of each of the four natural nucleotides. Unfortunately, these functional groups are excellent nucleophiles and can thus react with any electrophilic center that is in close proximity (reviewed in [47]). In general, these reactions can lead to inappropriate modifica- tions that alter the hydrogen-bonding capabilities and in turn can dramatically affect proper base-pairing during DNA replication to enhance the overall extent of mutagenesis. It is beyond the scope of this review to critically evaluate all the identified forms of DNA damage. However, one interesting example is O6-methylguanine, a miscoding DNA lesion that can be induced by

a variety of alkylating agents including methyl methanesulfonate [48] and tobacco-related carcinogens such as 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone [49] and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanol [50]. Alkylation at the O6-position of guanine changes the hydrogen bonding potential of the nucleobase through modification to the keto oxygen. This alteration changes the tautomeric form of the base from guanine to a form that resembles adenine and thus changes the Watson-Crick hydrogen bonding pattern of guanine from a d d * * to (-)*a*d (Fig. 11). The net result is that O6-methylguanine is poten- Fig. (10). Structure of the Klentaq DNA polymerase. tially miscoding. Indeed, it is generally accepted that the preferential Regardless of these differences, there is relative consensus with incorporation of dTTP opposite the lesion results from more stable 6 6 respect to the role of the fingers domain during polymerization. The base-pairing of the T:O -methylguanine pair as opposed to the C:O - structures of several DNA polymerases complexed with nucleic acid methylguanine pair. This, however, does not appear to be the case 6 in the absence and presence of dNTP reveal features consistent with since melting studies of DNA duplexes containing O -methylguanine 6 the mechanism described in Fig. 9 determined through kinetic stud- reveal that C:O -methylguanine is actually more energetically stable 6 ies. In these models, the dNTP binds to the polymerase:DNA com- than T:O -methylguanine [51]. Additionally, NMR studies indicate 6 plex in a template-independent manner through interactions with the that T:O -methylguanine contains fewer hydrogen bonding interac- 6 fingers domain. This initial binding event is followed by a conforma- tions than C:O -methylguanine [52,53] and support the supposition tional change that corresponds to rotations of the fingers domain that that the kinetically favored mispair is actually thermodynamically then induces "tighter" binding with the templating nucleobase. One less stable. hypothesis is that this rotation provides the driving force to align the incoming dNTP with its complementary partner and/or to orient the 6 3’-hydroxyl of the primer for attack on the bound dNTP. The kinetic behavior of nucleotide incorporation opposite O - methylguanine is likewise more complicated than predicted if one

Thymine H3C O N O CH3

N N HN 6 Guanine O -Methylguanine dR N N H C N O 3 NH2 O dR N O N NH N H C N N 3 Cytosine dR H N NH2 dR N N O 2 NH2 N N N dR N N NH2 O dR Fig. (11). Base-pairing capabilities of O6-methylguanine. 153 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 247 relies solely on strict hydrogen-bonding recognition patterns. For and angles relative to a Watson-Crick base pair. Thus, it was pre- example, Spratt and Levy [54] reported using the exonuclease- dicted that these bases would not be recognized as "damaged" DNA, deficient form of the E. coli Klenow fragment that the catalytic effi- an important feature required for the propagation of any novel base ciency for dTTP incorporation is 3-fold higher than that for the incor- pair within the cell. poration of dCTP. Surprisingly, the preference for dTTP does not Guanine H H arise through an effect on nucleotide binding as expected since the Iso-Guanine O H N N H O Km for dTTP and dCTP are essentially identical at ~100 μM. Instead, N N the higher catalytic efficiency reflects the faster kcat value measured N NH N N NH N for dTTP incorporation. N N N dR N dR O H N dR Other DNA polymerases such as Taq and Tth are similar to the E. NHO dR Cytosine H coli Klenow fragment as they preferentially incorporate dTTP oppo- H Iso-Cytosine site O6-methylguanine [54]. However, the underlying mechanism for this preferential insertion differs with these polymerases as the ~10- Iso-Guanine fold higher catalytic efficiency for dTTP incorporation arises from (enol tautomer) H both a 3-fold faster kcat as well as a 3.5-fold enhancement in binding O CH3 N affinity for dTTP compared to dCTP [54]. The mechanism of transle- N H sion synthesis catalyzed by the bacteriophage T4 DNA polymerase is N HN surprisingly different than these aforementioned polymerases. Un- N N N published data reveals that the polymerase preferentially incorporates dR OH O dR dTTP opposite the lesion rather than dCTP. However, it is interesting Thymine that the binding of dCTP to the T4 polymerase is favored ~20-fold compared to the binding of dTTP (A. Berdis, unpublished data). The Fig. (12). Structural comparison of non-natural base pairs of iso-guanine and iso-cytosine with the natural base pair guanine:cytosine. perturbation in binding affinity for dTTP is offset by a significant increase in the polymerization rate constant for incorporation in Using the E. coli Klenow fragment, Benner’s group demonstrated which the kpol using dTTP is nearly 100-fold faster than using dCTP that the iso-cytosine: iso-guanine base pair is indeed formed [63]. (A. Berdis, unpublished data). Unfortunately, the overall fidelity of this new base pair is low since deoxy-isoguanine can be easily incorporated opposite a templating Collectively, these kinetic data indicate that incorporation oppo- thymine and vice versa [62]. The facile incorporation of dTTP oppo- site miscoding lesions is influenced by perturbations in binding affin- site deoxy-isoguanine reflects the fact that the un-natural nucleotide ity that result from the loss of proper hydrogen bonding interactions can easily tautomerize from the expected keto to the enol form (Fig. as well as by alterations in the rate constants for incorporation. How- 12). Thus, the lack of intrinsic selectivity negated the use of deoxy- ever, the magnitude of these effects as well as how they contribute to iso-guanine to create a "third base pair". the overall fidelity of replication are polymerase-dependent. The absence of a universal mechanism for misincorporation opposite a Attempts to improve fidelity were made using 5-(2,4-diamino- miscoding DNA lesion such as O6-methylguanine suggests that hy- pyrimidine) and xanthosine to form a new base pair that resembles a drogen-bonding interactions are not used universally nor are they G:C base pair but that was different with respect to hydrogen bonding used equally by all DNA polymerases. arrangements in the minor groove (Fig. 13). It was predicted that these new minor groove interactions would enhance fidelity for form- ATTEMPTS TO CREATE "NEW" HYDROGEN BONDING INTERAC- ing the un-natural base pair. Indeed, the E. coli Klenow fragment was TIONS. Much effort has been placed in trying to understand the mo- able to incorporate the deoxyxanthosine triphosphate opposite 5-(2,4- lecular basis of genetic diseases caused by mutagenesis by studying diaminopyrimidine) with relatively high efficiency [64]. In addition, how DNA polymerases "behave" when they encounter various DNA this base pair possesses a high degree of fidelity since the deoxy- lesions. While these studies are of great medical importance, it is xanthosine triphosphate is poorly incorporated opposite natural clear that the ability of polymerases to effectively initiate and propa- pyrimidines, T and C. Unfortunately, the increase in fidelity does not gate mistakes can be utilized for applied biochemical research to unequivocally correlate with an absolute improvement in polymeriza- rationally re-design nucleic acid. One such example is the attempt to tion efficiency as the Klenow fragment could not incorporate the expand nature's genetic code to introduce a new "third" base pair triphosphate form of deoxyribose 5-(2,4-diaminopyrimidine) opposite (reviewed in [55]). The expansion of the genetic code from four po- a templating xanthine. Thus, these base pairs represent an example of tential pairing combinations to eight would enable the encoding of asymmetric polymerization as the polymerase shows unequal effi- additional information for the synthesis of novel proteins containing ciency for forming one base pair as compared to its complement. The an array of un-natural amino acids [55]. This technology would ulti- underlying mechanism for this "asymmetry" in DNA polymerization mately expand the repetoire of functional groups present on the is not clearly understood at this time. However, this phenomenon amino acid side chains to increase the stability of proteins [56] or prohibited attempts to fully replicate this novel base pair. The issue of expand the chemical diversity of existing enzymes [57]. In addition, asymmetric DNA polymerization will be a recurrent theme that hin- the development of novel base-pairing partners would create new ders efforts in the development of novel base pairs and the expansion biological polymers that could acts as universal primers for polym- of the genetic code. erase chain reaction (PCR) [58], novel catalysts [59], biosensors [60], H and nanowires [61]. Guanine Xanthine H O H N O H N The first published work directed towards rationally designing a N N N "third base-pair" was reported by Steve Benner's group [62]. These N N N NH NH efforts initially focused on using unique arrangements of hydrogen N N dR N N H N bonding acceptor-donor pairs to construct base pairs that are geomet- NHO dR dR H O dR rically identical to existing Watson-Crick base pairs. Of several pos- H Cytosine H sible combinations, only two potential base pairs were qualitatively 5-(2,4 diaminopyrimidine) evaluated for their ability to be formed enzymatically. These base pairs include iso-cytosine:iso-guanine (Fig. 12) and 5-(2,4-diamino- Fig. (13). Structural comparison of non-natural base pair of xanthine and 5- pyrimidine:xanthosine base pairs (Fig. 13). Despite the different ar- (2,4 diaminopyrimidine) with the natural base pair guanine:cytosine. rangements of hydrogen-bond acceptor-donor pairs, each novel base Regardless of these pitfalls, other groups have tried to overcome pair is predicted to maintain the proper interglycosyl bond distance difficulties associated with fidelity by developing analogs with

154 248 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis unique hydrogen bonding patterns. For example, Rappaport used a strategy emphasizing the contributions of steric guidance in addition similar design strategy to develop a novel base pair with 5-methyl-2- to hydrogen-bonding patterns to generate novel base pairs [67]. This pyrimidinone and 6-thioguanine (Fig. 14) [65]. He demonstrated that unique design would retain Watson-Crick hydrogen-bonding interac- the Klenow fragment could efficiently incorporate 5-methyl-2- tions between the two nucleobases to optimize their association. pyrimidinone opposite templating 6-thioguanine and vice versa [64]. However, the introduction of bulky side groups devoid of hydrogen- Furthermore, the efficiency of incorporation and extension was re- bonding interactions would facilitate discrimination against forming ported to be almost equivalent to that of forming a natural C:G base mispairs with naturally occurring nucleobases (A, C, G, and T). An pair [65]. Unfortunately, the 5-methyl-2-pyrimidinone:6-thioguanine example of this design is the formation of the pyridin-2-one:2-amino- base pair suffers from a lack of fidelity since dCTP is also easily 6-(N,N-dimethylamino)purine base pair [68]. As shown in Fig. 15, incorporated opposite 6-thioguanine while dGTP is also effectively the heterocyclic nitrogen and keto group of pyridin-2-one can form inserted opposite 5-methyl-2-pyrimidinone. Watson Crick hydrogen bonds with the complementary heterocyclic 6-Thioguanine nitrogen and exocyclic amino group present on 2-amino-6-(N,N- 2-Thiopurine dimethylamino)purine. The presence of the 6-dimethyl-amino group N S CH3 N S CH3 provides enough steric hindrance to prevent interactions with either N NH N thymine or cytosine. However, this group would not hinder associa- N NH N N N tion with pyridin-2-one since this analog lacks a functional group at dR O N NH2 dR dR N O dR this position. Consistent with this strategy, the Klenow fragment effi- 5-methyl-2-pyrimidinone 5-methyl-2-pyrimidinone ciently incorporates the pyridin-2-one analog opposite the un-natural purine [68]. Despite this rational design, selectivity is again a major Guanine 2-Thiopurine complication as dTTP is incorporated opposite 2-amino-6-(N,N- CH N O 3 N S H2N dimethylamino)purine with an efficiency comparable to that of pyri- NH N din-2-one [68]. N N NH N N N N To combat the issue of non-selectivity, the 6-dimethylamino dR NH O dR N dR O dR group was replaced with 6-(2-thienyl) [69]. The introduction of this H Cytosine 5-methyl-2-pyrimidinone bulkier group would prevent association with natural pyrimidines Fig. (14). Structures of base pairing interactions between 6-thioguanine and 5- (Fig. 16). As predicted, this modification enhances selectivity as the methyl-2-pyrimidinone, guanine and 5-methyl-2-pyrimidinone, 2-thiopurine pyridin-2-one analog is incorporated opposite 2-amino-6-(2-thienyl) and 5-methyl-2-pyrimidinone, and 2-thioguanine and cytosine. purine more efficiently than the misincorporation of any natural nu- cleotide [69]. Specifically, the K for pyridin-2-one incorporation Attempts to improve the fidelity of 5-methyl-2-pyrimidinone m opposite 2-amino-(6-thienyl)purine is 170 μM and the V is re- 2 max were made by using 2-thiopurine instead of 6-thioguanine [66]. As ported to be 11%/min . This represents a mere 10-fold reduction illustrated in Fig. 14, 2-thiopurine should effectively pair with 5- compared to normal DNA synthesis. Furthermore, the incorporation methyl-2-pyrimidinone but not with thymine or cytidine due to steric of dCTP and dTTP opposite 2-amino-6-(2-thienyl)purine are hin- hindrance imposed by the sulfur group. The fidelity of replicating the dered compared to the incorporation of pyridin-2-one. 5-methyl-2-pyrimidinone:2-thiopurine pair was evaluated using the wild-type T7 DNA polymerase that is proficient in exonuclease The molecular reason for this discrimination is noteworthy since proofreading capabilities [66]. The goal of this study was to evaluate it differs from what was predicted to occur based upon the molecular the efficiency of net nucleotide incorporation of various natural and determinants of 2-amino-6-(2-thienyl)purine. For example, it was un-natural nucleotides by measuring their incorporation and excision predicted that discrimination against dTTP incorporation would be opposite normal or modified templating nucleobases. Rappaport achieved through significant alterations in binding affinity since the demonstrated that the efficiency of net incorporation for the 5- keto oxygen should sterically clash with the 6-(2-thienyl) group. Sur- methyl-2-pyrimidinone:2-thiopurine pair is only 10-fold less than that prisingly, this is not the case as discrimination occurs primarily for replicating the normal adenine:thymine base pair [66]. This study through by a 4-fold reduction in the Vmax for incorporation rather than represents the first demonstration of maintaining a relatively high a perturbation in binding affinity. In contrast, dCTP incorporation degree of replication fidelity in the presence of exonuclease proof- opposite 2-amino-6-(2-thienyl)purine is hindered through a more reading by using a mixture of natural, un-natural, and non-natural pronounced effect on ground state binding. In this case, the Km of 430 nucleotide analogs. μM for dCTP is significantly larger than the Km values of 170 μM and 270 M measured for pyridin-2-one and dTTP, respectively. 2-Amino-6-(N,N-dimethylamino)-purine μ Adenine Although dCTP binds weakly, it is surprising that the Vmax for the H3CCH3 H H N incorporation of this nucleotide opposite 2-amino-(6-thienyl-purine) N N O CH3 HN is actually faster than that measured for pyridin-2-one incorporation N N N HN (compare 18 vs 11 %/min, respectively) [69]. These examples again N O dR N N N N N H reiterate the difficulty in using hydrogen-bonding interactions as a dR O dR dR H predictor to achieve discrimination against forming certain base pairs. Thymine Pyridin-2-one Replacement of the S with O also has interesting effects on the dynamics of incorporation that are not predicted by simple hydrogen 2-Amino-6-(N,N-dimethylamino)-purine Adenine bonding interactions or steric constraints. In general, the substitution O CH3 of an oxygen for sulfur has little effect on binding affinity since iden- H3CCH3 H H N N N tical Km values are measured for the incorporation of pyridin-2-one, N HN dCTP, and dTTP [70]. However, this substitution creates a measur- N N N N N able effect on the V of the reaction in which the value for the 2- O dR max N N N H dR N amino-(6-furanylpurine) analog is ~4-fold faster than the Vmax meas- O dR dR H ured for the 2-amino-(6-thienyl-purine) analog. Thymine Pyridin-2-one Fig. (15). Structures of the 2-amino-6-(N,N-dimethylamino)purine:pyridin-2- one base pair. 2 HYDROGEN BONDING AND STERIC GUIDANCE. In an attempt to The Vmax values reported here reflect the amount of primer elongation as a function of improve fidelity, Yokoyama et al. were amongst the first to develop a time for the non-natural nucleotide as compared to that for the incorporation of a natural dNTP opposite a natural templating nucleobase. 155 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 249

2-Amino-6-(thienyl-purine) 2-Amino-6-(thienyl-purine) this prediction, Taq DNA polymerase showed an unexpectedly strong bias toward incorporating dATP opposite several of the non-natural S H N S 2 nucleotides despite the fact that predicted hydrogen-bonding interac- HN N N N tions did not provide evidence for this selectivity [72]. One notable N N N O dR exception is 1,2-pyrazole-3-carboxamide. This analog meets the crite- N N H dR N Pyridin-2-one N N N H O ria of a universal base since dCTP and dTTP are incorporated with dR H dR H Cytidine equal frequencies [72]. Despite this promiscuity, however, it should 2-Amino-(furanylpurine) be noted that the overall catalytic efficiency for the incorporation of dCTP opposite 1,2-pyrazole-3-carboxamide is 10-fold lower than O dCTP incorporation opposite a template guanine. In these cases, the HN N N reduced catalytic efficiency arises from significant perturbations in O dR the binding affinity of the incoming nucleotide rather than an effect N N H N on Vmax values. H dR Pyridin-2-one Subsequent attempts to optimize the ambiguity of this analog Fig. (16). Base pairing interactions of 2-amino-(6-thienyl)purine with pyridin- have focused on manipulating the electronic features of the analog 2-one versus cytosine. through permutations in the number and location of heterocyclic ni- TAKING ADVANTAGE OF PROMISCUOUS BASE PAIRS. These exam- trogens. In this design, the pyrazole-3-carboxamide was further ples emphasize the dichotomy observed using alterations in hydro- changed to a class of triazoles that include (1H)-1,2,3-triazole-4- gen-bonding interactions as the predominant molecular determinant carboxamide, (2H)-1,2,3-triazole-4-carboxamide, and 1,2,4-triazole- to design a novel base pair. In general, DNA polymerases can incor- 4-carboxamide (Fig. 18) [75]. All three analogs are rather promiscu- porate these analogs effectively opposite their designed cognate part- ous in their ability to act as templating base for multiple incoming ner. However, the major obstacle associated with this design is the dNTPs. Of these three analogs, the (1H)-1,2,3-triazole-4-carboxamide difficulty in attaining selectivity for replicating the novel base pair. In displays the highest degree of selectivity as the catalytic efficiency for nearly all of the aforementioned examples, the major complication incorporating dGTP is 10-fold higher than dATP and greater than 50- was unforeseen promiscuity in base-pairing interactions as the un- fold more efficient than the incorporation of the pyrimidines dCTP or natural nucleobase can base pair with one or more natural nucleo- dTTP [75]. In contrast, the 1,2,4-triazole-3-carboxamide and (2H)- tides. 1,2,3-triazole-4-carboxamide are “universal bases” as the catalytic efficiencies for the incorporation of various dNTPs are nearly identi- The lack of fidelity exhibited by various un-natural nucleotides cal. In particular, (2H)-1,2,3-triazole-4-carboxamide is the most pro- has lead to the development of "universal" nucleobases, i.e., nucleo- miscuous as nearly identical Vmax/Km values are measured for the tide analogs that do not discriminate amongst natural bases [71]. The incorporation of dATP, dCTP, and dTTP. In contrast, identical ability to pair with multiple partners allows these analogs to over- Vmax/Km values for the incorporation of dCTP and dTTP are meas- come ambiguities arising from the degeneracy of the genetic code, ured opposite 1,2,4-triazole-3-carboxamide. Despite the observed and thus have applications toward nucleic acid biochemistry includ- promiscuity, the catalytic efficiency for incorporation opposite either ing utilization as PCR primers, DNA sequencing reagents, interfer- triazole is significantly lower than that measured using 1,2-pyrazole- ence RNA, and oligonucleotide probes for mutation diagnostics and 3-carboxamide. The reduction arises from higher Km values rather microorganism detection/differentiation. than large perturbations in Vmax values. These kinetic differences Amongst the earliest candidates as “universal bases” are the vari- argue that changes in the electronic configuration of the nucleotide ous azole carboxamide derivatives (Fig. 17) developed by Bergstrom influences ground-state binding rather than the phosphoryl transfer and Davisson [72-74]. These compounds are ambiguous by virtue of step. rotation around the amide bond that presents two alternative hydro- More recent attempts to achieve promiscuity have focused on gen-bonding patterns. Although each non-natural base is similar in oxadiazole carboxamides, a series of C-linked nucleoside analogs that present heteroatoms (nitrogen or oxygen) on the minor and major O O sides of DNA [76] Two noteworthy analogs are 1,2,4-oxadiazole-5- NH C 2 C NH2 O N NH2 N N C O N C NH2 R R N pyrrole-3-carboxamide 1,2-pyrazole-3-carboxamide N N N O O N R C NH2 C NH2 R N (1H)-1,2,3-triazole-4-carboxamide 1,2,4-triazole-3-carboxamide N O N N C NH2 R R NN imidazole-3-carboxamide 1,5-pyrazole-3-carboxamide Fig. (17). Structures of various pyrrole and pyrrazole nucleotide analogs. N shape and size, they differ with respect to composition. These differ- R (2H)-1,2,3-triazole-4-carboxamide ences provide each nucleobase with a unique electronic signature [74] that is proposed to influence their ability to pair with various dNTPs. Fig. (18). Structures of various triazole carboxamide nucleotide analogs. The ability of these analogs to allow degenerate replication was first carboxamide and 1,2,4-oxadiazole-3-carboxamide (Fig. 19). Kinetic examined by measuring the ability of Taq DNA polymerase to per- studies reveal that both oxadiazole analogs can be effectively incor- form PCR using a DNA template containing each azole derivative porated opposite T or G [76]. However, the placement of the heteroa- [72]. Since each pyrmidine-like analog presents two alternative hy- toms plays an unexpected role as the overall catalytic efficiency for drogen-bonding patterns, it was predicted that the purines, dATP and 1,2,4-oxadiazole-3-carboxamide is ~4-fold higher than that for the dGTP, would be inserted with nearly equal efficiencies. Contrary to 156 250 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

1,2,4-oxadiazole-5-carboxamide derivative. The higher catalytic effi- in nearly ever instance reported thus far, the major obstacle toward ciency derives from a 5-fold lower Km value for the 3-carboxamide creating a new base pair is maintaining fidelity. In most case, there is analog compared to the 5-carboxamide derivative. There is no overt indiscriminate pairing between natural and non-natural nucleotides. difference in the Vmax for either analog opposite C or T. This feature Finally, there still exist significant difficulties in the ability of DNA argues that the relative positioning of the heteroatoms plays a role in polymerases to effectively elongate beyond the incorporated non- dNTP recognition. However, it is currently unknown if this effect is natural nucleobase. This problem may reflect the lack of or change in caused by alterations in hydrogen bonding interactions or through location of critical hydrogen-bonding groups in the minor groove of changes in the electronic configuration of each analog. DNA. In is interesting to note that the oxadiazole analogs developed by Davisson’s group are one of the few analogs that show promise in O O NH circumventing this problem. C NH2 C 2 O N BIOMEDICAL APPLICATIONS OF UNIVERSAL NUCLEOBASES. De- N N O N spite limitations in rationally designing a new, specific base pair, the promiscuity displayed by certain analogs does have potential applica- R R tions in the development of novel chemotherapeutic agents. One im- 1,2,4-oxadiazole-5-carboxamide 1,2,4-oxadiazole-3-carboxamide portant area is in the rational design of anti-viral agents that take ad- Fig. (19). Structures of oxadiazole carboxamides that act as "universal" base vantage of low fidelity replication catalyzed by most viruses. Viral analogs. DNA synthesis is viewed as an error-prone process since at least one When tested for extension, it was demonstrated that Taq polym- mutation is introduced per round of replication [78]. The low but erase poorly extends beyond 1,2,4-triazole-3-carboxamide when appreciable mutation rate is beneficial for many viruses since this paired opposite G or T. In fact, the overall catalytic efficiency for lack of replicative fidelity creates a heterogeneous virus population elongation is comparable to that measured for the polymerase extend- that produces diversity. In fact, this diversity is believed to generate ing a natural mismatch such as A:A or G:T [76]. In contrast, Taq selective point mutations in critical proteins such as reverse tran- polymerase extends beyond either oxadiazole derivative when paired scriptase and viral protease that are targets for anti-viral agents (re- opposite any natural nucleotide [76]. However, the highest catalytic viewed in [79]). In these examples, the creation of selective point efficiency for extension is measured when the non-natural analog is mutations is sometimes associated with the development of drug paired opposite G or T [76]. This final aspect is important as it pro- resistance to many anti-viral agents. While some mutations are toler- vides one of the first demonstrations that an ambiguous base pair can ated, too many may cause a loss of viral fitness and viability. This be efficiently formed and extended by a single DNA polymerase. phenomenon, referred to as error catastrophe, is an increase in muta- tion frequency beyond a critical threshold that supports viability of Another example of an unexpected ambivalent nucleobase is 4- the organism [80]. The concept of error catastrophe has biomedical methylpyrimid-2-one (Fig. 20) [77]. This pyrimidine analog was applications as it can be used to develop new anti-viral agents that actually designed to be a selective pairing partner of G since the 4- induce "lethal mutagenesis" [81,82]. The strategy of this approach is methyl group is expected to clash with the 6-amino group of adenine to use pro-mutagenic nucleotides so that the low fidelity viral polym- to hinder the incorporation of dATP. Although the 3-hydrogen of 4- erase will increase their mutation frequency beyond viability to in- methylpyrimid-2-one is predicted to collide with the 1-imino proton duce error catastrophe. of guanine, the interactions between the two bases are proposed to be favorable due to hydrogen-bonding interactions between the 2-keto NH2 group of 4-methylpyrimid-2-one with the 2-amino group of G. Ki- N OH netic data obtained using the E. coli Klenow fragment is consistent with this design since only dGTP is incorporated opposite the tem- O N plating 4-methylpyrimid-2-one [77]. However, the insertion of the HO triphosphate of 4-methylpyrimid-2-one shows unusual promiscuity as O it is incorporated opposite all four natural templating nucleobases [77]. It should be noted that purines are incorporated more efficiently OH than pyrimidines as the incorporation of dCTP and dTTP is hindered by significantly high K values (> 400 μM). Although dATP and Fig. (21). Structure of the pro-mutagenic nucleoside analog, 5-hydroxy- m deoxycytidine. dGTP have identical Km values of ~60 μM, the Vmax for dATP is ~4- H N fold faster than dGTP. Thus, it is quite surprising that the Klenow N O 2 fragment preferentially incorporates dATP opposite 4-methyl- N N H N pyrimid-2-one rather than dGTP, the designed partner for this non- N N natural nucleotide. O R H R N O CH3 H2N N N H H H NN N N H O N N R N N R O HN N N CH3 O N R R OR CH 3 O N N Fig. (22). Structure of Ribavarin and its base-pairing capabilities opposite cytidine or uracil. R O HN N N N R The effectiveness of this strategy was first demonstrated by the O Loeb laboratory using 5-hydroxydeoxycytidine (Fig. 21) as the pro- R H2N mutagenic nucleoside as it is capable of pairing with either guanine or Fig. (20). 4-methyl-pyridone pairing interactions opposite adenine or guanine. adenine [81]. Cell culture studies demonstrated that nearly 80% of the These examples emphasize the inherent difficulties in designing replicative potential of HIV was lost compared in the presence of 5- novel base pairs through permutations of hydrogen-bonding interac- hydroxydeoxcytidine compared to in the absence of the analog. Fur- tions as the predominant molecular determinant. On one hand, most thermore, sequence analysis of viral DNA revealed a disproportionate DNA polymerases can utilize these modified nucleotides with sur- increase in G to A substitutions that were predicted to occur from the prising efficiency to ultimately generate a non-natural pair. However, misincorporation of 5-hydroxydeoxytidine. Despite this success, 157 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 251 however, 5-hydroxydeoxycytidine has never been used clinically to hairpins that may result from the "self-pairing" capabilities of 5- combat HIV infections. nitroindole. This feature limits the use of these analogs as "universal" A more clinically relevant example of this strategy is the anti- primers for initiating DNA synthesis. viral activity exhibited by Ribavirin, the ribose form of 1,2,4-triazole- Despite the lack of hydrogen-bonding interactions, both 5- 3-carboxamide. As shown in Fig. 22, Ribavirin can ambiguously nitroindole and 3-nitropyrrole can be incorporated opposite any tem- base-pair with either cytidine or uracil/thymine by presenting differ- plating nucleobase as demonstrated through qualitative studies with ent hydrogen-bonding faces by rotation about the carboxamide group. the E. coli Klenow fragment [89]. However, once incorporated, each During the initial round of viral DNA synthesis, Ribavirin will be analog acted as a chain terminator as the polymerase is unable to incorporated opposite either C or U with equal efficiency. During extend beyond the non-natural nucleobase. It is quite surprising, how- subsequent rounds of replication, rCTP or UTP will then be inserted ever, that chain termination with the 5-nitroindole derivative occurred opposite any Ribavirin present in the template as a result of its misin- predominantly at purine sites [89]. This result implies that 5- corporation during the first round of replication. Thus, the pro- nitroindole is recognized and incorporated as a pyrimidine analog mutagenic nucleoside will promote viral mutagenesis to change the rather than as a purine analog. Although the mechanism underlying composition and sequence of the virus' genetic code to induce "error this unusual kinetic behavior is still under investigation, these results catastrophe". Although Ribavirin has been used as an anti-viral agent were amongst the first to demonstrate that polymerization can occur for many years [83], its mechanism of action remained elusive until in the complete absence of hydrogen bonding interactions. However, Crotty et al. [82] demonstrated that Ribavarin could be incorporated the major pitfall with nitro-containing nucleotides lies in their lack of opposite templating C and U with nearly identical catalytic efficien- selectivity as well as in their inability to be extended by most DNA cies. In addition, it was shown that rCTP and UTP could be incorpo- polymerases. rated opposite templating Ribavarin with equal efficiencies to main- ACTIVE SITE GEOMETRY AS A MOLECULAR FORCE FOR POLYM- tain an overall symmetry in the replication of this non-natural nu- ERIZATION. Arguably the most recognized example of efficient repli- cleobase. Finally, cell culture studies reveal that Ribavarin reduced cation in the absence of hydrogen-bonding interactions is the demon- infective poliovirus production to as little as 0.00001%. This reduc- stration by the Kool laboratory that 2,4-difluorotoluene (Fig. 24), the tion did not appear to reflect a simple inhibition of viral replication non-hydrogen-bonding isostere of dTTP, is effectively incorporated since the anti-viral activity of Ribavarin coincided with its ability to opposite A [90]. In fact, the overall catalytic efficiency for incorporat- induce mutations in the viral genome. The importance of this study is ing the non-natural nucleotide opposite adenine is only 100-fold that the cell culture data corroborate the kinetic data and thus vali- lower than that for forming a natural adenine:thymine base pair. At dates the pro-mutagenic effects of the non-natural nucleotide. This the molecular level, the Vmax for the incorporation of difluorotoluene result supports the concept of inducing error catastrophe to combat opposite A is only ~3-fold slower than that for the incorporation of viral replication. Equally important, this provides clear evidence that dTTP [90]. However, the K value for difluorotoluene opposite a pro-mutagenic nucleosides can be developed as anti-viral (and per- m templating A is 95 μM, a value that is ~40-fold higher than the Km for haps anti-microbial) agents to combat mutagenesis associated with dTTP [90]. Collectively, these data indicate that the shape of the ana- the development of drug resistance. log is important for the polymerization. However, one should not O O ignore the fact that hydrogen-bonding interactions play a significant N - role as manifest in the large perturbation in binding affinity caused by + O N - + O the lack of these functional groups. Despite the low catalytic efficiency, however, it is clear that 2,4- N N difluorotoluene is preferentially incorporated opposite A and is R R poorly incorporated opposite G, C, and T [90]. The low catalytic 3-nitro-pyrrole 5-nitroindole efficiency for non-natural nucleotide opposite these natural templat- ing nucleobases reflects perturbations in binding affinity in addition Fig. (23). Structures of 3-nitro-pyrrole and 5-nitroindole. to significant alterations in Vmax values. In fact, the selectivity for DEPARTURE FROM HYDROGEN-BONDING FUNCTIONAL GROUPS. incorporation opposite A arises from a 250 to 2,600-fold reduction in Attempts to alleviate the dependency of hydrogen bonding initially Vmax values. Another important feature is that the kinetic parameters lead to the development of 3-nitro-pyrrole [84] and 5-nitroindole for forming the 2,4-difluorotoluene:adenine pair differ from those [85]. In these cases, the presence of the nitro group removes classical measured for the formation of a thymine:guanine mismatch. In this hydrogen bonding interactions but also enhances stacking interactions case, the Vmax for forming a T:G mismatch is 10-fold slower than by polarization of the
-aromatic system of the pyrrole or indole ring forming the 2,4-difluorotoluene:adenine pair while the Km for dTTP system. Although modeling studies of 3-nitropyrrole predicted that opposite G is significantly higher at 580 μM. the non-natural nucleotide should pair with any templating nu- Adenine cleobase, the introduction of 3-nitropyrrole in duplex DNA actually o H H leads to a significant destabilization of 14 C [84]. The reason for this N N O CH3 large destabilization effect became evident from structural studies indicating that the nitro group protrudes into the major groove [86] N N HN N and thus does not stack as well as initially predicted. dR N O dR The purine analog, 5-nitroindole, is less destabilizing than 3- Thymine nitro-pyrrole, its pyrimidine counterpart [85]. Crystal structures of Adenine DNA containing 5-nitroindole reveal that this analog is involved in H H more favorable base-stacking interactions due to overlap with adja- N N F CH3 cent aromatic bases [87]. When tested for in vitro applications, the N introduction of a single 5-nitroindole residue into a primer for PCR or N DNA sequencing did allow for ambiguous pairing at its complemen- dR N F dR tary position and subsequent elongation of the modified primer [88]. However, the ability to prime DNA synthesis becomes significantly 2,4 Difluorotoluene worse when two or more 5-nitroindole residues are introduced into Fig. (24). Structural comparison of non-natural base pair of adenine: 2,4- the same primer [88]. The decrease in efficiency is caused by the difluorotoluene with the natural base pair adenine:thymine. ability of the oligonucleotide to form secondary structures such as

158 252 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

Collectively, these results clearly indicate that hydrogen-bonding a platform to rationally design novel base pairs based on shape and interactions between the incoming nucleotide and the templating size constraints rather than through hydrogen bonding potential. nucleobase are not needed for efficient polymerization. The more EXPLORATION OF ACTIVE SITE TIGHTNESS AS A MODEL FOR DNA provocative implication, however, is that the DNA polymerase does POLYMERIZATION. To test the model of active site geometry as well as not utilize hydrogen bonding interactions to achieve fidelity during to design new base pairs completely devoid of hydrogen bonding the selection of the correct nucleotide during the replication cycle. In interactions, the Kool laboratory designed 4-methylbenzimidazole as fact, one could argue that the hydrogen bonding potential of the form- a complementary pair for 2,4-difluorotoluene. As illustrated in Fig. ing base pair can only be used to discriminate against the formation 3 25, the base pair comprised of 4-methylbenzimidazole opposite of mismatches rather than for positive selection of proper base pair . difluorotoluene is proposed to be geometrically identical to an ade- Indeed, several examples will be provided later which demonstrate nine:thymine base pair. In this design, the methyl group of 4- that the lack of hydrogen bonding interactions can allow for facile methylbenzimidazole is roughly the same size as the amino group of incorporation. adenine and the fluoro groups of 2,4-difluorotoluene are similar in Another interesting and often overlooked result of these studies is size to the keto oxygen of dTTP. Kinetic studies reveal that the over- that the catalytic efficiency for the incorporation of 2,4- all efficiency for the incorporation of 2,4-difluorotoluene opposite 4- difluorotoluene opposite 2,4-difluorotoluene is higher than that methylbenzimidazole is 200-fold lower than that for the incorporation measured for incorporation opposite adenine, its predicted shape/size of dTTP opposite A [94]. In addition, the efficiency of forming the partner [90]. Although the Vmax for both incorporation events are non-natural base pair is greater than that for dTTP incorporation op- essentially identical, the Km value of 53 μM for 2,4-difluorotoluene posite 4-methylbenzimidazole. Surprisingly, the presence of a hydro- opposite itself is 2-fold lower than the value of 95 μM measured for gen-bonding group actually decreases the efficiency by perturbing incorporation opposite adenine. Therefore, while 2,4-difluorotoluene both the Km and Vmax values for the polymerization reaction. This is considered to be a selective partner for adenine, the non-natural result marks an important milestone in developing a third base pair as nucleotide is actually more inclined to form a "self pair" as it is pref- it demonstrates the potential to obtain selectivity without using hy- erentially incorporated opposite itself. The "self pairing" capabilities drogen-bonding interactions. of 2,4-difluorotoluene is not unique as it is observed with a number Adenine on nucleobases completely devoid of classical hydrogen bonding H H functional groups (vide infra). N N F CH3

Regardless of these issues, the incorporation of isosteric analogs N N of natural nucleotides such as 2,4-difluorotoluene into DNA indicates dR N that canonical Watson-Crick hydrogen bonds are not essential for F dR polymerization. As an alternative to these molecular forces, Kool has 2,4 Difluorotoluene proposed that the predominant force in optimizing polymerization 4-Methylbenzimidazole fidelity is the geometrical alignment of the incoming nucleobase with N CH3 F CH3 the template base [91]. The dynamics of this model are commonly referred to as "shape complementarity" or "steric fit" [92]. According N to this model, the ability of a polymerase to distinguish between the dR F correct dNTP versus other incorrect dNTP depends upon the size and dR geometry of DNA polymerase's active site [97]. In this model, the 2,4 Difluorotoluene active site of the polymerase is compared to a room containing a floor 9-Methylimidazo[(4,5)-b]pyridine (the flat side of the primer 3'-end base), a far wall (the templating N CH3 F CH3 base), and a ceiling (the space provided by the enzyme as it closes down over the incoming nucleotide) [92]. The floor and ceiling of the N N active site are proposed to be "fixed" with respect to the shape and dR F size required to accommodate all four nucleotides. However, the dR depth and shape of the far wall will obviously vary since the size of 2,4 Difluorotoluene the templating base varies as a consequence of differences in base Fig. (25). Comparison of several mixed, non-natural base pairs including composition, i.e., pyrimidines versus purines. Therefore, in order for adenine: 2,4-difluoro-toluene, 4-methylbenzimidazole: difluorotoluene, and 9- an incoming nucleotide to be processed properly, it must fit into the methylimidazo[(4,5)-b]pyridine: 2,4-difluorotoluene. consensus shape defined by the active site which also takes into ac- Unfortunately, two major pitfalls are associated with this base count interactions present along the major and minor grooves of DNA pair and thus limit its utility. The first complication again lies in the [92]. lack of symmetry during replication. Although 4-methylbenzimida- This model invoking geometrical constraints and size exclusion zole is incorporated opposite template difluorotoluene, the reaction is has generated significant interest in the field of DNA polymerization far less favorable than the incorporation of 2,4-diflurotoulene oppo- for several reasons. First, it provides a viable model to explain how site 4-methylbenzimidazole [94]. The second problem is the inability factors other than hydrogen bonding interactions are used for replica- of the 4-methylbenzimidazole:2,4-difluorotoluene base pair to be tion. This includes the formation of natural base-pairing partners as selectively extended [94]. Indeed, primer extension reactions demon- well as for the misreplication of miscoding DNA lesions such as O6- strate that a thymine:4-methylbenzimidazole mispair is extended methylguanine. Secondly, the facile incorporation of difluorotoluene more efficiently than the 2,4-difluorotoluene:4-methylbenzimidazole opened debate into which biophysical parameter is most important for pair. Likewise, the adenine: 2,4-difluorotoluene is extended more replication efficiency and DNA stability. These debates have been efficiently than the 4-methylbenzimidazole: 2,4-difluorotoluene pair. appropriately summarized in a recent review article by Kool and To optimize the replication of this base pair, 4-methylbenzimida- Sintim [93]. Briefly, seminal questions into the hydrogen-bonding zole was modified slightly to 9-methylimidazo[(4,5)-b]pyridine (de- potential of difluorotoluene as well the polar nature of this non- noted as dQ). This analog contains an additional heterocyclic nitrogen natural nucleotide are discussed. Finally, the steric fit model provides group that does not significantly alter its shape (Fig. 25) [95]. How- ever, the addition of this functional group as well as its position fac-

3 ing the minor groove of DNA was proposed to allow for facile exten- This type of mechanism in which hydrogen-bonding interactions are used for discrimina- sion [95]. The kinetic behavior of this new partner for 2,4- tion against the formation of replication errors rather than being used exclusively for the formation of proper base pairs has been dubbed the "negative selection" model of polym- difluorotoluene is similar to that of 4-methylbenzimidazole except erization (97). 159 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 253

O F Cl

H3C H3C H3C NH

N O F Cl HO HO HO O O O

OH OH OH

Thymine 2,4-Difluorotoluene-Deoxyriboside 2,4-Dichlorotoluene-Deoxyriboside

Br I

H3C H3C

Br I HO HO O O

OH OH 2,4-Dibromotoluene-Deoxyriboside 2,4-Diiodotoluene-Deoxyriboside Fig. (26). Structures of thymidine analogs that vary in size as a function of various substituent groups. that it is more selective as a template base [95]. In addition, dQTP is now fragment [97]. In fact, the low steric selectivity of <40 for Dpo4 inserted opposite a templating 2,4-difluorotoluene with an efficiency was proposed to result from the enzyme possessing a flexible or equal to that of dATP [95]. Finally, it was demonstrated that the E. "loose" active site that can adapt to multiple base-pair sizes rather coli Klenow fragment can extend dQ paired opposite 2,4- than a spatially large active site [97]. In addition, these data suggest difluorotoluene 300-fold more efficiently compared to when 4- that hydrogen-bonding interactions may be important in the overall methylbenzimidazole is paired opposite 2,4-difluorotoluene [94]. fidelity of this low fidelity DNA polymerase [97]. These results indicate that the purine N3 hydrogen bond acceptor A recent report by Sintim and Kool has also evaluated the influ- group plays an important role in polymerization fidelity as well as in ence of steric effects through modification of the shape of a base pair the kinetics of DNA elongation. by altering the size of the non-natural nucleotide at specific positions The importance of steric effects on nucleotide selection selectiv- in a molecule [98]. Using toluene as a nucleobase "skeleton", the ity have been further measured through kinetic studies that systemati- shape of the molecule was altered through systematically introducing cally evaluate the effects of base pair size [96-98]. The approach used combination of H, F, Cl, and Br atoms at variable positions. In gen- in these studies was to gradually increase the size of the base pair eral, large kinetic effects on the catalytic efficiency were obtained that using a series of nonpolar thymidine shape mimics (Fig. 26) through resulted from subtle changes in the size and location of the substituent the introduction of different halides at positions corresponding to 2- group [98]. It should be noted that while the Klenow fragment is and 4- of thymine. Steady-state rates of polymerization were first highly sensitive to small differences in nucleobase shape, a clear reported using the high fidelity bacteriophage T7 DNA polymerase as correlation between binding affinity and/or rate constants for polym- a model [96]. In these experiments, the base pair size was varied in erization with the shape of the formed base pair could not be deter- small increments of 0.25 Å. In addition, polymerization reactions mined. Regardless, the shape analogs in this series displayed a re- were performed monitoring the incorporation of natural nucleotides markable 3,500-fold range in enzymatic efficiency despite the fact opposite variably sized thymine analogs in the template and, con- that they are all closely related toluenes [98]. versely, for variably sized dTTP analogs opposite natural template Table 1. Different DNA Polymerases have Varying Requirements bases. In general, the high fidelity T7 polymerase displayed high for Efficient Nucleotide Insertion selectivity for a specific base pair size [96]. The efficiency in nucleo- tide incorporation decreased 280-fold for non-natural base pairs that are 0.4 Å larger than the optimum size of a natural pair (10.6 Å). Klenew Fragment Pol
Pol  Pairs smaller than 0.3 Å had corresponding reductions in catalytic efficiencies [96]. The data were interpreted with respect to active site O tightness as the primary determinant for high fidelity displayed by N NH replicative polymerases. Not Essential Not Essential Essential N The influence of steric effects within the active site of Dpo4, a Y- N NH2 family DNA polymerase, has also been examined using a similar approach [97]. It was proposed that this low-fidelity DNA polym- O erase is prone to making mistakes since its active site is more "open" N compared to high fidelity DNA polymerases [97]. This "open" active NH Not Essential Essential Essential site predicts that the enzyme has lower geometrical constraints for N N NH2 forming a base pair and should thus readily incorporate the nonpolar thymidine analogs opposite purines. Indeed, it was demonstrated that Dpo4 preferred to pair thymidine shape mimics with adenine [97]. In addition, the preferred size was at the median point for the range of With an arsenal of non-natural nucleotides in hand, the Kool base size (0.15 Å) that was also reported for the E. coli Klenow frag- laboratory has also tested the ability of DNA polymerases from dif- ment, a polymerase with higher fidelity compared to Dpo4. However, ferent polymerase families to incorporate and extend beyond various the size preference with Dpo4 was quite small compared to the Kle- non-natural nucleotides devoid of hydrogen bonding interactions. As expected, the kinetics of incorporation vary significantly amongst the 160 254 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis different DNA polymerases tested and thus indicate that the dynamics facilitate phosphoryl transfer. It is interesting that MMLV reverse of nucleobase recognition are not universal [99]. The results from transcriptase cannot incorporate non-natural nucleotides whereas HIV these studies are summarized in Table 1. Polymerases such as the E. reverse transcriptase readily uses a variety of non-natural analogs coli Klenow fragment and HIV reverse transcriptase could easily [99]. Taken together, these data suggest that the mechanism and dy- insert molecules devoid of any functional groups opposite natural or namics of nucleotide incorporation vary even amongst members of non-natural template bases. As a general rule, these polymerases do the same polymerase family. not appear to rely on the presence of hydrogen bonding and minor In contrast to MMLV reverse transcriptase, pol
and Klenow groove functional groups during nucleotide incorporation. A second fragment can efficiently utilize these nucleotide analogs [101,102]. In group of polymerases represented by the eukaryotic pol
could also some cases, the catalytic efficiencies for the incorporation of these insert molecules devoid of hydrogen bonding groups. However, they analogs are only 10-fold lower than that measured for forming a natu- could not incorporate analogs devoid of minor groove functional ral base pair. With pol
, the presence of the nitro group enhances the groups. Finally, a third group represented by pol  could not insert binding affinity for nucleotide as the Km values for 5-nitrobenzimida- molecules that lack functional groups needed for hydrogen bonding zole, 6-nitrobenzimidazole, and 5-nitroindole are lower than that or that play a role in minor groove interactions. These studies clearly measured for benzimidazole. In addition, the Km values for nitro con- indicate that DNA polymerases have different requirements for ca- taining analogs are only 10-fold higher than the binding affinities talysis and suggest that the mechanisms accounting for nucleotide measured for natural dNTPs. The opposite trend is observed with the incorporation and discrimination are not universal. Klenow fragment as the binding affinities for all non-natural nucleo- Benzimidazole 5-Nitrobenzimidazole tides are ~100-fold higher than those for the natural dNTPs. Despite N N NO2 these differential effects on binding affinity, the overall catalytic effi- ciencies using these non-natural analogs are 1,000-fold more favor- N N able than those measured for forming natural mispairs. This result is R R reminiscent of the incorporation of non-natural nucleotide, 2,4- 6-Nitrobenzimidazole 5-Nitroindole difluorotoluene, opposite adenine which possesses more favorable N NO2 kinetic parameters than those measured for forming natural mis- matches [90]. N NO2 N R Perhaps the most striking result of this study is the lack of shape R complementarity observed during the incorporation of the various Fig. (27). Structures of benzimidazole, 6-nitrobenzimidazole, 5- nitrobenzimidazoles opposite their predicted partners [101,102]. Ac- nitrobenzimidazole, and 5-nitroindole. cording to the shape complementarity model, these benzimidazole In addition to monitoring incorporation kinetics, the Kool labora- analogs should be preferentially incorporated opposite C and T. tory has also critically evaluated the ability of various DNA polym- However, in nearly all cases examined, the Klenow fragment prefer- erases to extend beyond nucleobases devoid of hydrogen bonding entially inserted these non-natural purine analogs opposite natural functional groups [100]. Their studies identified two general classes purines rather than pyrimidines. Likewise, pol
also incorporates of polymerases that differ in their ability to elongate beyond non- some nitrobenzimidazoles opposite A and G with nearly identical natural base pairs. Polymerases such as the E. coli Klenow fragment, catalytic efficiencies compared to their incorporation opposite the Taq DNA polymerase, and HIV reverse transcriptase could extend pyrimidines, C and T. Although these data indicate that hydrogen beyond nonpolar base pairs containing 9-methylimidazo[(4,5)- bonding interactions are not needed to form a base pair, they also b]pyridine. However, these polymerases were unable to extend be- indicate that the "steric fit" model is not universal. Thus, the ability to yond 4-methylbenzimidazole, the closely related analog. The differ- form a "correctly" shaped base pair is not an absolute requirement for ence in extension kinetics suggests that these polymerases utilize a nucleotide incorporation. specific minor groove interaction in order to elongate DNA. Other These data were used to propose an alternative model for DNA polymerases including the T7 DNA polymerase as well as the eu- polymerization that invokes "negative selection" of inappropriate karyotic enzymes, pol and pol , could not elongate any base pair
 nucleotides as the predominant force used to maintain replication containing a nonpolar, non-hydrogen bonding nucleobase. At face fidelity [101]. The negative selection model postulates that during the value, these data suggest that these polymerases absolutely require binding of dNTPs, the polymerase allows the nucleobase of the dNTP the presence of hydrogen bonding groups that participate in minor and the template base to adopt the lowest free energy conformation groove interactions. Alternatively, it is possible that these polym- during their interaction. If the interactions are favorable, the polym- erases are sensitive to perturbations in DNA structure that are likely erase then allows the conformational change to occur to facilitate to occur as a consequence of these non-natural nucleobases. Collec- phosphoryl transfer. However, incorrect pairing prevents the confor- tively, these data argue that the requirements for hydrogen-bonding mational change and thus does not allow catalysis to occur. Through functional groups for primer elongation are also not universal. this mechanism, the polymerase can sample a wide variety of nucleo- "NEGATIVE SELECTION" AS A MODEL FOR NUCLEOTIDE INCOR- tides. However, only the dNTP that adopts the lowest free energy PORATION. To further delineate the underlying mechanism of nucleo- conformation will be accepted due to the formation of favorable in- tide selection, the Kuchta laboratory has measured the incorporation teractions. This model differs from most positive selection models of several purine analogs such as benzimidazole, 5- that postulate that the polymerase is allowed to sample only a limited nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole (Fig. 27) number of possibly correct partners. [101]. The kinetics of nucleotide incorporation were measured using Another interesting example of divergent mechanisms in nucleo- three different polymerases that include the eukaryotic pol , the E.
tide selection is that exhibited by human DNA primase [103]. The coli Klenow fragment, and the Moloney-murine leukemia virus role of DNA primase is to synthesize short RNA primers needed for (MMLV) reverse transcriptase. MMLV reverse transcriptase was the initiation of Okazaki fragment synthesis. Primase is arguably one unable to incorporate any of the non-natural nucleotides opposite of the most error-prone polymerases as it easily misincorporates natu- templating nucleobases or an abasic site, a non-templating DNA le- ral rNTPs to generate a series of natural mismatches [104]. Surpris- sion [101]. The lack of incorporation apparently reflects the inability ingly, this polymerase is unable to utilize non-natural nucleotides of the reverse transcriptase to catalyze phosphoryl transfer since the such as 4- and 7-(trifluoromethyl)benzimidazole or 7- -D-guanine non-natural nucleotides can prevent the binding of natural dNTPs to  that resemble natural nucleobases with respect to shape/size and hy- the polymerase to inhibit replication [101]. These results suggest that drophobicity yet that are devoid of proper hydrogen bonding interac- the MMLV reverse transcriptase does not require hydrogen-bonding tions [103]. In fact, primase is only able to incorporate rNTP analogs groups for nucleotide binding but instead utilizes these interactions to 161 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 255 that contain hydrogen bonding functional groups [103]. These results is advantageous for several reasons. First, the nucleotides should not indicate that human DNA primase requires the formation of hydrogen suffer from a lack of fidelity induced by the effects of tautomerization bonding interactions between the incoming nucleotide and templating that were observed with base pairs such as iso-cytosine and iso- nucleobase in order for polymerization to occur. Primase is not guanine [63]. In addition, the hydrophobic interactions between the unique in this requirement as several other polymerases including bases are predicted to be strong and selective for forming hydropho- AMV reverse transcriptase and MMLV reverse transcriptase are also bic pairs. Finally, it is argued that mispairs between the non-natural resistant toward incorporating nucleotides devoid of hydrogen- and natural bases will be disfavored since the energetic cost for bonding capabilities [105]. This is interesting as each of these polym- desolvation of a natural base will be substantially higher than that for erase is considered to be either a low-fidelity or error-prone DNA a non-natural nucleotide [108]. polymerase, and one would predict that they would utilize these natu- 7-Azaindole ral nucleotides.

NH2 N NH2 N N N O CH3 N N dR O N HN dR N N N dR N N Isocarbostyril dR O dR Fig. (29). Structures of 7-azaindole and isocarbostyril nucleosides. Of several potential base pairs examined, the pair formed be- NH tween 7-azaindole and isocarbostyril nucleosides possesses notewor- NH H2N N NH N thy thermodynamic and kinetic properties. Despite the lack of hydro- NH N gen bonding interactions, the 7-azaindole:isocarbostyril pair is only N N N slightly less stable than an adenine:thymine base pair ( T~2oC) N N
dR dR O [111]. Equally important, the incorporation of isocarbostyril triphos- dR phate opposite 7-azaindole is very efficient as evident in a kcat/Km Fig. (28). The tautomerization of adenine allows for pair with dTTP or dCTP. value of 2.7*105 M-1min-1 [111], which is only 100-fold lower than The correlation between low-fidelity and the requirement for the incorporation of dTTP opposite adenine. In addition, replication hydrogen bonding interactions for polymerization has intriguing of the non-natural base pair appears to be kinetically symmetrical as ramifications for the underlying mechanism of the DNA replication. the kcat/Km value for the incorporation of 7-azaindole triphosphate A speculative model is that low fidelity DNA polymerases have opposite isocarbostyril is 1.8*105 M-1min-1 and nearly identical to the evolved to rely almost exclusively on hydrogen bonding interactions value of 2.7*105 M-1min-1 measured for the incorporation of isocar- between the incoming dNTP and the templating base. These hydro- bostyril opposite 7-azaindole [111]. Although the overall catalytic gen bonding patterns can be used to stabilize the association of the efficiencies for forming the 7-azaindole:isocarbostyril is low com- incoming nucleotide with a templating partner. However, tautomeri- pared to that for a natural base pair, the catalytic efficiency for form- zation can change the hydrogen bonding potential of either the in- ing a mispair with natural dNTPs is ~100-fold less favorable. This coming nucleotide and/or the templating base to decrease polymeriza- increase in replication fidelity is consistent with the overall design of tion fidelity. An example of this is illustrated in Fig. 28 in which ade- using hydrophobicity as the driving force for polymerization. nine can interconvert between the amino and an imino form. The Unfortunately, the enzymatic formation of self-pairs consisting of amino form of adenine will specifically pair with thymine while the 7-azaindole:7-azaindole and isocarbostyril:isocarbostyril are essen- imino form will preferentially pair with cytosine. The possibility of tially identical to that for forming 7-azaindole:isocarbostyril [111]. such a mechanism has been proposed by several groups using bio- Thus, three new base pair combinations were inadvertently created chemical [63] or theoretical [106] approaches. rather than a single base pair as originally intended. The 7-azaindole EVALUATING HYDROPHOBICITY AS A MOLECULAR DETERMINANT self-pair is formed enzymatically with reasonable efficiency as it is FOR FIDELITY. Arguably the most under-appreciated biophysical only 200-fold slower than the formation of natural base pairs [111]. force during DNA polymerization is desolvation. Goodman and Surprisingly, the fidelity for forming the mispair is also rather high as Petruska were amongst the first to critically evaluate the role of natural dNTPs are incorporated poorly opposite the non-natural nu- desolvation and nucleobase hydrophobicity as the primary determi- cleobase. For example, the misinsertion of dATP opposite 7- nant for maintaining fidelity and enhancing the efficiency of incorpo- azaindole is the most favorable as it is 40-fold slower [111]. ration [107]. Indeed, close inspection of the helical nature of duplex Another pitfall of this system lies in the inability of these self DNA makes it intuitively obvious that desolvation plays a role during pairs to be easily extended. For example, the 7-azaindole:7-azaindole replication since the removal of water surrounding the functional self pair is poorly extended by the E. coli Klenow fragment [112]. groups of the incoming dNTP must occur prior to the formation of Surprisingly, mammalian polymerase  can extend the 7-azaindole:7- hydrogen bonds within the interior of the DNA helix. Until recently, azaindole as efficiently and selectively as a natural base pair [112]. it has been nearly impossible to unambiguously evaluate the role of This is remarkable sine polymerase  cannot synthesize the self pair desolvation as a mechanism of polymerase fidelity. This obstacle [112]. Taking advantage of this unique behavior, the Romesberg occurs since altering the functional groups of a natural nucleotide group used a binary polymerase system consisting of the E. coli Kle- generally affects the hydrogen-bonding potential and tautomeric form now fragment and eukaryotic polymerase  to enzymatically form of the nucleotide. Thus, changing one biophysical feature inadver- and extend the self-pair to synthesize full length DNA containing 7- tently influences another. azaindole in the template strand [112]. This represents another impor- The most innovative approaches toward studying the influence of tant milestone toward expanding the genetic code as this work dem- desolvation come from the collective studies of Romesberg and onstrates the feasibility of forming and extending a non-natural with Schultz. Their efforts represent rational attempts to generate a new both efficiency and fidelity. base pair using the hydrophobic nucleotides displayed in Fig. 29 THE MECHANISM AND DYNAMICS OF TRANSLESION DNA SYN- [108]. The overall design of these novel base pairs is based on princi- THESIS. Translesion DNA synthesis is the ability of a DNA polym- ples of hydrophobic packing originally derived from studies involv- erase to incorporate a nucleotide opposite damaged DNA and extend ing protein structure and stability (reviewed in [109,110]) and does beyond the damaged site to propagate a potential genetic mistake. not extensively rely on hydrogen-bonding potential or shape- Perhaps the most prevalent and pro-mutagenic class of DNA lesion is complementary. The lack of reliance on hydrogen bonding potential an abasic site which is considered to be the prototypical non-coding 162 256 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

O N NH

N Abasic site N NH2 O O O O O OH OH H Depurination O O O O - PO O PO O- PO O-

O O O

Tetrahydrofuran

O O OH

O

PO O-

O

Fig. (30). Structure of an abasic site and tetrahydrofuran moiety designed to functional mimic the non-templating DNA lesion.

4 DNA lesion (Fig. 30) . Abasic sites are formed by the hydrolysis of Vmax for incorporation opposite the abasic site is significantly lower the bond between the C1' of ribose and the N9 of a purine or the N1 than that for normal DNA replication. of a pyrimidine. Although abasic sites arise spontaneously under An important feature of these studies was the quantitative evalua- normal physiological conditions [113], their formation is enhanced by tion of nearest neighbor effects on the dynamics of nucleotide selec- exposure to ionizing radiation and certain chemicals [114] as well as tion [117]. With pol
, the V for dATP incorporation varies as the through the action of DNA glycosylases which act to excise damaged max penultimate base is changed. In fact, the highest Vmax value is meas- nucleobases [115]. Most abasic sites are repaired by the base excision ured with T as the penultimate base while the lowest is observed with repair pathway [116]. However, a small fraction of lesions can persist C [117]. At face value, these results suggest that the preferential in- and be replicated to yield a high probability of mutagenesis. Since no corporation of dATP arises through hydrogen-bonding interaction coding information is present at an abasic site, there exists a potential with the nearby templating base. However, this is not accurate since to incorporate all four dNTPs with equal efficiency. This situation the Vmax for dGTP incorporation opposite an abasic site is also high- could result in a 75% chance of incorporating the wrong nucleotide to est when T is the penultimate base rather than the C. cause a genetic mutation. Alternatively, the lack of coding informa- tion could provide a strong roadblock and prohibit movement of the Using the E. coli DNA polymerase I, Livneh's group reported that DNA polymerase to stop DNA replication completely. dATP is also preferentially incorporated opposite an abasic site [119]. In this case, the Km for dATP is 32 μM [119] and is only 3-fold worse INCORPORATION OF NATURAL NUCLEOTIDES OPPOSITE AN ABASIC than the Kd of 10 μM measured for the incorporation of dATP oppo- SITE. Despite the lack of coding information at an abasic site, DNA -1 site T [123]. The kcat value of 0.059 s for dATP opposite an abasic polymerases such as eukaryotic pol
[117] and pol  [118], the E. site is likewise reduced 850-fold compared to the k of 50 sec-1 coli DNA polymerase I [119], the bacteriophage T4 DNA polymerase pol measured for dATP opposite T [123]. Similar to pol
, the E. coli [120], and HIV reverse transcriptase [121] preferentially insert dAMP polymerase preferentially utilizes purines as opposed to pyrimidines. (and dGMP to a lesser extent) opposite an abasic site. This kinetic In general, the K values for purines are ~5-fold lower than those phenomenon is commonly referred to as the "A-rule" of translesion m measured for dCTP or dTTP while the kcat for dATP is faster than DNA synthesis (reviewed in [122]). The mechanism for the "A-rule" that for the other three natural nucleotides. is enigmatic since the ability of a polymerase to preferentially incor- porate one specific nucleotide opposite this non-templating lesion Low fidelity polymerases have also been tested for their ability to cannot be reconciled by models invoking hydrogen-bonding and/or perform translesion DNA synthesis. Goodman demonstrated that HIV shape complementarity interactions. In an attempt to define this reverse transcriptase is similar to high fidelity polymerase as it also mechanism, the Goodman laboratory investigated the incorporation preferentially incorporates dATP opposite an abasic site [121]. With of natural dNTPs using pol
isolated from Drosophila [117]. Their this enzyme, the Vmax/Km value for dATP incorporation opposite the kinetic analyses revealed that amongst the four natural nucleotides, abasic site is 5-fold higher than for the insertion of dGTP and 15- and dATP is incorporated the fastest, and is 5-fold faster than the Vmax 20-fold higher than dTTP and dCTP, respectively [121]. However, using dGTP [117]. In addition, the Km for dATP is 1.4 mM while that the preferential incorporation of dATP opposite the abasic site is not for dGTP is 3.4 mM, and these values are significantly lower than universal since certain error-prone DNA polymerases prefer to incor- those measured for the pyrimidines, dCTP and dTTP [117]. Of porate other natural nucleotides. For example, pol iota preferentially course, these values are significantly different that those measured incorporates dGTP opposite this lesion [124]. In fact, the polymerase during normal DNA synthesis. Specifically, the Km value for a correct appears to discriminate against dATP incorporation as dGTP and dNTP opposite its complementary partner is ~10-fold lower than that dTTP are incorporated with higher efficiencies than dATP [124]. measured for incorporation opposite an abasic site. Likewise, the Another polymerase that defies the "A-rule" is the Rev1 polymerase from yeast that preferentially incorporates dCTP opposite the abasic site [125]. These examples appear to reflect extreme behavior as both 4The vast majority of kinetic studies use a tetrahydrofuran as a stable mimic of an abasic polymerases show a distinct preference for utilizing one specific site. In most cases, the kinetic parameters for incorporation either lesion are indistinguish- dNTP. In the case of Rev 1, the polymerase also incorporates dCTP able between the two forms of damaged DNA. 163 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 257 opposite other DNA lesions that are not predicted to be the comple- preferential insertion of dATP opposite an abasic site could reflect the mentary partner of cytidine [126]. influence of desolvation. Our studies using the bacteriophage T4 DNA polymerase argua- An alternative model is the base-stacking model that is based bly provide the most comprehensive evaluation of the underlying upon the correlation between the measured kpol/Kd values of dNTPs mechanism of nucleotide selection during translesion DNA synthesis with their relative conformation when placed opposite an abasic site catalyzed by a high fidelity polymerase. Using single turnover reac- [120,127,128]. NMR studies indicate that dAMP exists in a thermo- tion conditions (enzyme in molar excess versus DNA substrate), we dynamically favored intrahelical conformation when placed opposite were able to define the kinetic dissociation constant, Kd, as well the the lesion [127]. This favored conformation correlates well with the maximal polymerization rate, kpol, for all four natural dNTPs opposite relative high kpol/Kd value for dAMP insertion. On the other hand, an abasic site [115]. As summarized in Table 2, dATP is preferen- dCMP and dTMP are found to exist in an unfavorable extrahelical tially incorporated compared to other dNTPs. This enhancement position [128] that coincides with their overall poor kpol/Kd values. arises in part due to the fact that dATP binds with higher affinity. In These observations led to us to propose that the efficiency of nucleo- this case, the Kd value of 35 μM for dATP is 4- to 34-fold lower than tide insertion directly reflects the “stacking” capabilities of the nu- the other natural nucleotides. In addition, the kpol value for dATP is cleobase [120]. -1 0.15 sec and is between 3- to 10-fold faster than that measured for The dynamics of this model were tested by correlating the ability the other dNTPs. of the T4 DNA polymerase to extend beyond an abasic site with the Table 2. Kinetic Parameters for dNTP Incorporation Opposite an proposed helical position of the nucleobase paired opposite the lesion. Abasic Site It was proposed that an intrahelical base such as adenine should be extended much more efficiently than an extrahelical base such as -1 dNTP Kd (μM) kpol (sec ) cytosine or thymine [120]. As predicted, the rate of extension from adenine paired opposite an abasic site is 20-fold faster than extension dATP 35 +/- 5 0.15 +/- 0.01 beyond a G:abasic site mispair and at least 100-fold faster than exten- dCTP 250 +/- 50 0.010 +/- 0.001 sion beyond a C:abasic mispair. Collectively, these data argue that the dGTP 130 +/- 20 0.02 +/- 0.01 efficiency of extension beyond a dNMP:abasic site is modulated by whether the terminal base pair exists in an intra- or extrahelical con- dTTP 1200 +/- 200 0.06 +/- 0.01 formation. Structural studies of the bacteriophage T4 homologue Values taken from [120]. poised at an abasic site provide further evidence for this model as adenine is observed in a thermodynamically favored conformation Using these values, the catalytic efficiency for the incorporation inside the helical structure of duplex DNA [129]. of dATP across from an abasic site is calculated to be 4.3*103 M-1s-1. This value is significantly lower than that of 107 M-1s-1 measured for THE USE OF NON-NATURAL NUCLEOTIDE TO PROBE SHAPE COM- the correct incorporation of dATP opposite T [17]. Surprisingly, the PLEMENTARITY DURING TRANSLESION DNA SYNTHESIS. Several labo- decrease in catalytic efficiency for translesion synthesis arises primar- ratories have used non-natural nucleotides as probes to further study ily from a reduction in the rate of the conformational change (~700- the dynamics of translesion DNA synthesis. One of the earliest en- fold) and only a minimal change in ground state binding (~ 3-fold). deavors in this field is work by the Kool laboratory that investigated Another significant difference in the dynamics of incorporation re- the role of shape complementarity during replication opposite an flects alterations in the time course in product formation measured abasic site [130]. The shape complementarity model predicts that under pseudo-first order reaction conditions (DNA and nucleotide in nucleotides possessing the correct shape and size of a "true" base pair molar excess versus polymerase). Using unmodified DNA, the time should be incorporated with higher catalytic efficiencies than those course for dATP incorporation opposite T is biphasic and indicates lacking these requirements. Indeed, the validity of model has been that a step after phosphoryl transfer is rate-limiting for polymerase strengthened by the demonstration that pyrene triphosphate (Fig. 31) turnover [17]. In contrast, the time course for dATP incorporation is inserted opposite an abasic site at least 100-fold more efficiently opposite the abasic site is linear, and indicates that phosphoryl trans- than any of the four natural dNTPs [130]. Using the E. coli Klenow fer or a step prior to that phosphoryl transfer is the rate-limiting step fragment, Morales and Kool demonstrated that the Km of 3 μM for pyrene triphosphate incorporation opposite the lesion is 27-fold better [120]. Furthermore, the lack of a significant elemental effect when
- than the Km of 85 μM measured for dATP [130]. Likewise, the kcat of S-dATP was substituted for
-O-dATP suggests that the phosphoryl -1 transfer step is not rate-limiting for incorporation opposite the DNA 9 sec for pyrene triphosphate is 10-fold faster than that measured for lesion [120]. Thus, the data from several independent experiments the incorporation of dATP [130]. The facile insertion of pyrene indicate that the conformational change step preceding phosphoryl triphosphate opposite the abasic site suggests that the "void" could be transfer limits the overall rate of nucleotide incorporation opposite an easily filled by the bulky aromatic nucleobase. Indeed, modeling abasic site. studies revealed that the overall shape and size of the pyrene:abasic site mispair is nearly identical to that of natural adenine:thymine pair MECHANISTIC DEDUCTIONS FROM KINETIC STUDIES. The studies [130]. with the bacteriophage T4 DNA polymerase revealed that the larger purines are inserted with an overall higher catalytic efficiency (kpol/Kd) compared to the smaller pyrimidines [120]. At face value, the higher catalytic efficiency for the larger nucleotides is consistent with the shape complementarity model proposed by Kool's group O O O [92]. However, there exist other alternatives including the effects of nucleobase desolvation and base-stacking capabilities rather than the P-O PO OPO O absolute size and/or shape of the molecule. As mentioned earlier with O- O- O- Romesberg's work [108], the solvation energies associated with non- OH natural nucleotides can dramatically influence the activity of a DNA 5 Fig. (31). Structure of pyrene triphosphate. polymerase. Since adenine has a relative low solvation energy , the The lack of hydrogen-bonding potential of either the abasic site or the incoming pyrene are consistent with the shape complementar- 5The solvation energy for adenine is -19.258 kcal/mol while that for cytidine, guanine, and thymine are - ity model. However, other factors such as base-stacking and desolva- 23.211 kcal/mol, -26.009 kcal/mol, and -11.903 kcal/mol, respectively. It should be noted that while thymine has the lowest solvation energy amongst the four natural nucleotides, it possesses the worst tion could also play a significant role in the incorporation of pyrene catalytic efficiency for incorporation opposite an abasic site. This argues that desolvation alone does not triphosphate opposite this DNA lesion. In fact, it is quite interesting dictate the efficiency of translesion DNA synthesis. 164 258 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

NH 2 O N N F NH N+ 2 O– N N N N N N

dR dR dR dR dR dATP IndTP 5-FITP 5-AITP 5-NITP

N NN

dR dR dR 5-CHITP 5-CEITP 5-PhITP

O O O P-O PO OPO dR = O O- O- O-

OH Fig. (32). Comparison of the structures of dATP with various 5-substituted indolyl deoxynucleotides. that pyrene triphosphate is incorporated opposite templating nu- To address the influence of these molecular forces, analogs corre- cleobase only 100-fold less efficiently compared to incorporation sponding to 5-fluoro-, 5-amino-, 5-cyclohexyl, 5-cyclohexene, and 5- opposite an abasic site [130]. The lower efficiency is caused by re- phenyl-indole-2’deoxyriboside triphosphates (Fig. 32) were synthe- ductions in Vmax which are ~1% that measured for incorporation op- sized and characterized for incorporation opposite an abasic site posite the lesion. Surprisingly, the Km for pyrene triphosphate is es- [133,134]. Kd and kpol values for their incorporation are summarized sentially identical (>10 μM) regardless of templating nucleobase in Table 3. The 5-fluoro- and 5-amino- analogs are similar to the [130]. Thus, while the alterations in Vmax values can be explained by indole derivative as all three are poorly inserted opposite the abasic simple steric exclusion, the ability of the polymerase to bind the site [133]. The high Kd values of 150-200 μM and the low kpol values large, bulky nucleotide rather tightly regardless of shape complemen- of ~0.2 sec-1 for these analogs arguably reflect the loss of energetic tarity argues that other physicochemical parameters may influence its stabilization caused by the lack of -electrons present at each sub- incorporation. stituent group. THE ROLE
-ELECTRON SURFACE AREA DURING TRANSLESION The most interesting trend is observed for the incorporation of 5- DNA SYNTHESIS. We further evaluated the contributions of shape cyclohexyl, 5-cyclohexene, and 5-phenyl-indole-2’deoxyriboside complementarity, base-stacking, and solvation energies toward nu- triphosphates. The bacteriophage T4 DNA polymerase incorporates cleotide incorporation by measuring the insertion of a series of modi- the 5-phenyl derivative opposite an abasic site with an extremely high -1 fied nucleotides opposite an abasic site. Our initial studies identified a catalytic efficiency. The kpol of 53 sec and Kd of 14 μM are nearly unique non-natural nucleotide, 5-nitro-indolyl-2’-deoxyriboside identical to those measured for the 5-nitro analog [131,133] and sug- triphosphate (5-NITP) (Fig. 32), that was inserted opposite an abasic gests that analogs containing conjugated functional groups bind to the site with approximately 1,000-fold greater efficiency compared to polymerase with high affinity and are inserted faster opposite the that for dAMP insertion [131]. Kinetic characterization reveals that 5- abasic site. NITP is incorporated opposite an abasic site with a fast kpol value of -1 Table 3. Kinetic Parameters for the Incorporation of 5-Substituted 126 sec and a low Kd value of 18 μM. Remarkably, these values are Indolyl Nucleotides Opposite an Abasic Site nearly identical to those for the enzymatic formation of a natural Watson-Crick base pair [17] and reiterate the finding that hydrogen dXTP K ( M) k (sec-1) bonding potential is not absolutely essential for nucleotide insertion d μ pol [70,85,90]. Although 5-NITP is also inserted opposite natural nu- 5-NITP a 18 +/- 3 126 +/- 7 cleobases with varying degrees [131], the catalytic efficiency is 30- b IndTP 145 +/- 10 0.28 +/- 0.07 fold greater for translesion DNA synthesis. The high catalytic effi- c ciency results from an enhancement in the conformational change 5-FITP 152 +/- 41 0.30 +/- 0.03 c step preceding chemistry (high kpol value) rather than an enhancement 5-AITP 255 +/- 40 0.17 +/- 0.01 in binding affinity (identical Kd values). 5-PhITP c 14 +/- 3 53 +/- 4 The importance of the nitro moiety was first evaluated by simply 5-CEITP d 5.1 +/- 1.7 25 +/- 2 replacing this functional group with -H [132]. This replacement re- 5-CHITP d 44 +/- 14 0.70 +/- 0.13 duces the catalytic efficiency for nucleoside insertion by ~2,300-fold aValues taken from [131]. b Values taken from [132]. c Values taken from [133]. d Values taken from [134]. [132]. Although binding affinity is perturbed 8-fold, the major effect of this substitution is the 450-fold reduction in the rate of the confor- mational change step that corresponds to a change in relative free To test this mechanism, the isosteric analogs, 5-cyclohexyl and 5- cyclohexene, were tested for incorporation opposite an abasic site energy (

G) of 3.62 kcal/mol. This energetic difference is proposed to arise through base-stacking interactions mediated between the [134]. The catalytic efficiency for the 5-cyclohexene-indole analog is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The overlapping -electron densities of the conjugated indole nucleoside higher efficiency reflects a substantial increase in the kpol value (com- with the polymerase and DNA. However, other forces such as desol- -1 vation as well as size and shape complementarity cannot be unambi- pare 25 versus 0.7 sec , respectively) rather than an influence on guously refuted based solely upon these data. ground-state binding. The faster kpol value for the 5-cyclohexene- 165 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 259

Fig. (33). Proposed model for DNA polymerization that invokes the contributions of
-electron interactions. indole derivative indicates that
-electron density enhances the rate of change step required to place the templating base in an intrahelical the enzymatic conformational change step required for insertion op- conformation that then allows for proper alignment of the primer- posite the abasic site. However, the near identity in Kd values sug- template required for the phosphoryl transfer step (kchem) (Fig. 33, gests that biophysical parameters independent of
-electron density step C). At an abasic site, the rate of this conformational change is such as desolvation and/or size also influence dNTP binding. significantly increased since the lack of a templating nucleobase cir- cumvents the need for this re-positioning. Indeed, it is notable that the AN ALTERNATIVE MODEL FOR DNA POLYMERIZATION. The re- sults of the kinetic analyses were interpreted with respect to the non-natural nucleotides are poorly incorporated opposite templating physical properties of each nucleotide analog in conjunction with nucleobase [131-134]. This result argues that the rate of this confor- available structural information of various DNA polymerases. An mational change step is slowed when a templating base is present. essential caveat of this model is that the templating nucleobase is This most likely is caused by the shear bulk of the non-natural nu- oriented in a conformation that prevents direct hydrogen-bonding cleotide hindering the facile re-positioning of the templating nu- contacts between the templating base and the incoming nucleotide cleobase from an extra- into an intra-helical conformation. 6 during the initial binding event [135-138] . Since the orientation of It should be emphasized that this model differs from the shape the templating nucleobase is in an extrahelical position, this creates a complementarity model for several reasons. First, the shape comple- transient "void" in the DNA that provides a functional mimic of an mentarity predicts that large non-natural nucleotides such as 5-phenyl abasic site (Fig. 33, step A). It is easy to envision that large, bulky and 5-cyclohexyl-indoles should both be effectively inserted opposite nucleotides such as 5-phenyl and 5-cyclohexyl-indoles can easily fill the abasic site since both provide the proper size and shape to ade- the "void" produced by the transiently-formed abasic site intermedi- quately fill the void. Despite having similarities in shape and size, ate. Since both molecules are similar in shape and size, their KD val- however, the kpol value for the cyclohexyl analog is ~100-fold slower ues are essentially identical and independent of templating nu- than that for the cyclohexene analog. The most discernible difference cleobase. Unlike binding affinity, however, the kpol value is highly between these two analogs is with respect to
-electron density. Thus, dependent upon the presence of a templating nucleobase. We argue while dNTP binding opposite the DNA lesion appears to be influence that the kpol step (Fig. 33, step B) represents the conformational by shape/size constraints in addition to the hydrophobicity of the incoming nucleotide, the rate of the conformational change is directly linked with the presence of
-electron density at the 5 position of the 6This supposition is supported by the structures of several DNA polymerases [130-132] modified indole. At the atomic level, we propose that this stabiliza- that provide physical evidence precluding direct Watson-Crick pairing interactions due to tion is mediated through interactions with aromatic amino acids pre- the expected orientation of the template. 166 260 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis sent in the active site of the DNA polymerase [131]. The enzyme- are very similar (compare 1.6 to 1.1 s-1 for normal versus translesion mediated conformational change transfers the aromatic nucleobase DNA synthesis, respectively) while the Kd for dATP opposite dam- into the aromatic environment of the duplex DNA and provides en- aged DNA is only 4-fold higher than that for non-damaged DNA tropic stabilization. The dynamics of this model are currently being (compare 27 versus 110 μM, respectively) [150]. The implication of examined through site-directed mutagenesis studies coupled with these kinetic data is that error prone DNA polymerases have a diffi- structural investigations. cult time discriminating between damaged and non-damaged DNA. This suggests that the coding information present at the bulky lesion DNA SYNTHESIS OPPOSITE BULKY DNA LESIONS. Another well- characterized DNA lesion is the thymine dimer which is comprised of is used to guide nucleotide incorporation. Thus, the bulky lesion is two adjacent and covalently-bonded thymines (Fig. 34). The forma- not processed as a "transient" abasic site as proposed for the T7 DNA tion of this lesion is catalyzed predominantly by ultraviolet radiation polymerase [146]. This model is supported by the crystallographic [140]. This lesion can be directly repaired by DNA photolyase [141] evidence of yeast pol
bound to a thymine dimer [151] which dem- or via the nucleotide excision repair pathway [145]. Unfortunately, onstrate that the thymine dimer is positioned in an intrahelical posi- replication of unrepaired DNA lesions occurs frequently to produce tion. This contradicts data obtained with the T7 DNA polymerase characteristic mutations in dipyrimidine sequences that represent [148] showing that the lesion is placed in an extrahelical position. initiation events in carcinogenesis. For example, squamous cell carci- These differences suggest that error prone polymerase such as yeast noma involves mutations in the p53 gene [143] while basal cell carci- pol
have "loose" active sites in which the thymine dimer remains noma [144] and melanoma [145] involve mutations in the PATCHED inside the helix during replication. In contrast, high fidelity DNA gene and the p16 gene, respectively. polymerase such as the bacteriophage T7 enzyme have a "tight" ac- tive site that makes its necessary for the bulky lesion to exist in an Using the T7 DNA polymerase as a model, the Taylor laboratory extrahelical position outside the polymerase's active site. provided kinetic evidence that a thymine dimer is processed as a non- templating lesion rather than a miscoding DNA lesion [146]. Their Non-natural nucleotides have again been used to probe the dy- works demonstrated that dATP was preferentially inserted versus namics of these mechanisms. Specifically, the Taylor laboratory has dGTP followed by dCTP and dTTP. The pattern of nucleotide incor- compared the kinetic parameters for the incorporation of pyrene poration is similar to that observed for incorporation opposite an triphosphate opposite a thymine dimer using the high fidelity T7 abasic site. In addition, the selectivity for dATP incorporation oppo- DNA polymerase or the error prone pol
[147,152]. With the T7 site the 3’-T of the thymine dimer is 50-fold higher than that for DNA polymerase, the pyrene triphosphate is incorporated opposite dGTP7. The degree of incorporation selectivity opposite 3'-T matches the 3’-T of the thymine dimer 15-fold more efficiently than dATP that exhibited during replication opposite an abasic site [147]. This [147]. This degree of selectivity is less than that of ~58 observed for feature suggests that both lesions are processed via an identical the incorporation of pyrene triphosphate opposite an abasic site. mechanism. It was thus proposed that the 3‘-T of the DNA lesion However, it was quite striking that the pyrene triphosphate is poorly exists in an extrahelical position and functionally mimics an abasic incorporated opposite the 5’-T of the thymine dimer. In fact, the se- site [146]. In contrast, the 5’-T is held tightly and remains in an intra- lectivity for pyrene triphosphate is remarkably low at <0.01. Thus, the helical position so that it is processed as a templating base [146]. The difference in selectivity for the non-natural nucleotide at the 3’-T dynamics of this model are supported by crystallographic evidence of versus the 5'-T of thymine dimer led to the proposal that part of the the T7 DNA polymerase bound to a thymine dimer reported by the lesion is placed in a extrahelical position to mimic an abasic site Ellenberger laboratory [148]. In this structure, the thymine dimer lies [147]. The error-prone polymerase yeast pol
showed more signifi- outside the helical structure of the DNA to form a cavity resembling cant differences in the selectivity for incorporating the pyrene an abasic site. triphosphate opposite either DNA lesion [152]. During replication opposite a thymine dimer, there was a 170-fold preference for pyrene O O O O CH3 CH3 triphosphate versus dATP and a larger, 3,600-fold increase for inser- CH3 CH3 UV radiation HN HN HN NH tion opposite the 3’-T of a thymine dimer [152]. At face value, it would appear that these differences in selectivity provide evidence O N O N O NNO HH that a thymine dimer is not replicated like an abasic site. However, it was pointed out by the author's of this work that the specificity of the Two adjacent thymine residues cis, syn thymine dimer non-natural nucleotide is largely dependent upon the templating base and secondary to the primer-template [152]. This observation can be Fig. (34). Structure of a thymine dimer. explained by a mechanism in which the non-natural nucleotide inter- There is convincing biological data that thymine dimers can also calates between the templating bases and/or displaces the templating be replicated by various error-prone DNA polymerases. For example, strand so that it is now placed in an extrahelical conformation [152]. the Goodman laboratory reported that polymerase V from E. coli Future work is needed to provide more definitive answers as to how preferentially incorporates dATP opposite the bulky lesion [149]. this bulky lesion is "properly" replicated by error-prone polymerases This result is similar to that reported for the T7 DNA polymerase and and misreplicated by high fidelity enzymes. Toward this goal, we are suggests that the dynamics of replication for high and low fidelity currently applying our series of non-natural nucleotides to understand polymerases are identical. However, this is not entirely accurate as how the high fidelity T4 polymerase behaves when it encounters a the degree of nucleotide selectivity differ significantly between the thymine dimer. Preliminary data with the high fidelity T4 DNA po- two polymerase systems and argues that the mechanisms are in fact lymerase indicate that analogs that are efficiently incorporated oppo- distinct [149]. In particular, the E. coli polymerase V incorporates site an abasic site such as 5-NITP and 5-PhITP are not effectively dATP ~50-fold more favorably than dGTP during replication oppo- incorporated opposite the thymine dimer (B. Devadoss and A. Berdis, site a thymine dimer [149]. However, the selectivity for dATP versus unpublished data). For example, 5-NITP is rapidly inserted opposite -1 dGTP is only 2-fold when the polymerase performs translesion DNA an abasic site with a kpol value of 126 sec [131]. However, the incor- synthesis opposite an abasic site [149]. Similar differences in kinetic poration of 5-NITP opposite a thymine dimer is reduced 6,000-fold behavior have also been reported by the Prakash laboratory using pol (B. Devadoss and A. Berdis, unpublished data). This difference sug-
from yeast as the model system [150]. Surprisingly, pol
replicates gests that the T4 DNA polymerase does not replicate a thymine dimer a thymine dimer with a similar catalytic efficiency compared to non- through the formation of an abasic-site intermediate as proposed for damaged DNA [150]. In this case, the polymerization rate constants the T7 DNA polymerase [147]. However, a detailed kinetic study is needed to unambiguously validate this conclusion. 7The selectivity for incorporating dATP at the 5'-T is reported to be greater than 100 CONCLUSIONS AND FUTURE DIRECTIONS. DNA is a fascinating compared to dGTP [141]. This indicates that this portion of bulky lesion is replicated as a molecule that functions as the carrier of the genetic information "normal" thymine. 167 Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity Current Chemical Biology, 2007, Vol. 1, No. 3 261 needed for RNA transcription and subsequent protein translation. The non-natural base pair while another DNA polymerase is used for the ability of various enzymes to accurately replicate DNA represents one elongation step [112,160]. Another innovative strategy involves the of the most fundamental yet complex biological processes found in use of protein engineering to re-design DNA polymerases that can nature. In this review, we have attempted to succinctly describe vari- form and then elongate non-natural base pairs [161-163]. ous chemical approaches that have been used to probe the mechanism Another application of non-natural nucleotides lies in their ability and dynamics of this fascinating biological process. Several laborato- to be used to as building blocks for novel biopolymers in material ries have used a variety of nucleotide analogs to decipher the molecu- sciences. DNA (and RNA) are biopolymers that can mimic machine lar mechanism of DNA polymerization. The results of these laborato- functions such as tweezers [164], walkers [165], and gears [166]. In ries make it clear that hydrogen-bonding interactions are important addition, these new biopolymers can be used as ultrasensitive DNA for efficient and accurate DNA synthesis. However, these interactions detection sensors [167] or nanotransporter units [168] used in drug are not absolutely essential for replication to occur. In this regard, the release applications. Continuing to apply non-natural nucleotides to use of these analogs has lead to the development of several biophysi- these developing platforms will undoubtedly expand the repetoire and cal models that may explain the efficiency and fidelity of DNA syn- capabilities of these new and exciting application-based technologies. thesis. These models include the influence of steric fit/shape com- plementarity, negative selection, hydrophobicity/solvation energies, Finally, these analogs can be used in various biomedical applica- and
-electron surface area. Although each unique model is based tions. In this respect, we previously proposed a novel strategy to use upon convincing experimental evidence, it is unlikely that DNA po- selective non-natural nucleotide analogs to enhance the chemothera- lymerases utilize any one of these biophysical forces exclusively peutic effects of DNA damaging agents [169]. This proposal is based during DNA replication. Indeed, DNA polymerases have undoubt- on the demonstration that several of our indolyl nucleotides are in- edly evolved to use a combination of these molecular forces to corporated opposite an abasic site, a common form of DNA damage, achieve rate enhancement, selectivity, and fidelity. It is also clear that with higher efficiency than opposite templating DNA [133,169]. In these biophysical forces are not used equally amongst various DNA addition, the DNA polymerase is unable to extend beyond the incor- polymerases. For example, there is now significant evidence that high porated non-natural nucleotide, a result that provides direct evidence fidelity polymerases have a "tight" active site while low fidelity or for their unique chain termination capabilities [133,169]. The net error-prone polymerases have a "looser" active site that allows them result is that these nucleotide analogs can selectively inhibit transle- to make mistakes more easily. In addition, the high fidelity bacterio- sion DNA synthesis in vitro. Current efforts are currently underway phage T4 DNA polymerase appears to rely heavily on desolvation to test if these analogs can be used to enhance the chemotherapeutic and
-electron surface area while lower fidelity polymerases such as effects of DNA damaging agents by inhibiting pro-mutagenic DNA reverse transcriptase and human primase rely more heavily on hydro- replication. This inhibitory feature could be important in preventing gen-bonding interactions. Thus, a universal mechanism cannot be the development of drug resistance that is associated with mutagene- blindly applied to all DNA polymerases to explain their activity. This sis. feature has been carefully reviewed in a recent article by Joyce and Benkovic [153]. The article by Joyce and Benkovic points out that the ABBREVIATIONS existing kinetic data for correct versus incorrect nucleotide incorpora- dNTP = Deoxynucleoside triphosphate tion does not reveal a unified mechanism for fidelity [153]. We envi- sion that the various non-natural nucleotides described in this review A = Adenine will be used over the next several years to compare and contrast the C = Cytidine mechanism of action of different DNA polymerases. This work will G = Guanine undoubtedly provide further proof that polymerases behave differ- ently during correct and pro-mutagenic DNA synthesis. However, it T = Thymine will prove more interesting to see if polymerases that perform diverse dATP = Adenosine-2’-deoxyriboside triphosphate biological functions with the cell, i.e., genomic replication, DNA repair, and error-prone synthesis, employ different chemical and ki- dCTP = Cytosine-2’-deoxyriboside triphosphate netic strategies during DNA synthesis. dGTP = Guanosine-2’-deoxyriboside triphosphate In addition to understanding the basic mechanism of DNA po- dTTP = Thymine-2’-deoxyriboside triphosphate lymerization, the development of un-natural and non-natural nucleo- Q = 9-Methylimidazo[(4,5)-b]pyridine tides has had a significant impact on biotechnology. One clear exam- ple is in attempts to expand the genetic code to "create" a new, novel dQTP = 9-Methylimidazo[(4,5)-b]pyridine-2’-deoxyriboside base pair. Significant progress has been made by the Schultz [154- triphosphate 156] and Yokoyama [157-159] laboratories toward developing in 5-NITP = 5-Nitro-indolyl-2’-deoxyriboside triphosphate vitro and in vivo transcription/translation systems using non-natural base-pairs. Unfortunately, the full application of this methodology 5-NapITP = 5-Napthyl-indolyl-2’deoxyriboside triphosphate has been limited by barriers associated with the complete replication 5-PhITP = 5-Phenyl-indolyl-2’-deoxyriboside triphosphate of DNA containing non-natural nucleotides. There appears to be three 5-CE-ITP = 5-Cyclohexene-indolyl-2’deoxyriboside triphosphate distinct obstacles that currently hinder the utilization of un-natural and non-natural nucleotides as an artificial base pair. The first prob- 5-CH-ITP = 5-Cyclohexyl-indolyl-2’deoxyriboside triphosphate lem reflects the fact that most non-natural nucleotides are poor sub- strates compared to natural dNTPs. Indeed, the kinetic parameters for REFERENCES the non-natural analogs are typically 100-fold worse than those ob- [1] Knowles JR. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev served for forming a natural base pair. Secondly, many of these non- Biochem 1980; 49: 877-919. natural nucleotides are promiscuous such that there is an intrinsic lack [2] Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem 1999; 274: 17395-17398. of fidelity in forming a new base pair. In most cases, the non-natural [3] Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends nucleotide has multiple pairing partners and this hinders its ability to on the DnaB:DnaC ratio. Mol Microbiol 1995; 17: 825-831. be selectively replicated. Finally, most polymerases are unable to [4] Johnson RE, Prakash L, Prakash S. Biochemical evidence for the require- efficiently extend beyond a non-natural base pair. This deficiency ment of Hoogsteen base pairing for replication by human DNA polymerase iota. Proc Natl Acad Sci USA 2005; 102:10466-10471. obviously prohibits their complete replication. However, systems are [5] Yoshida K, Tosaka A, Kamiya H, et al. Arg660Ser mutation in Thermus being developed that use two distinct DNA polymerases to overcome aquaticus DNA polymerase I suppresses T-->C transitions: implication of this obstacle. In these cases, one DNA polymerase is used to form the wobble base pair formation at the nucleotide incorporation step. Nucleic Ac- ids Res 2001; 29: 4206-4214. 168 262 Current Chemical Biology, 2007, Vol. 1, No. 3 Devadoss and Berdis

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Received: October 10, 2006 Revised: November 20, 2006 Accepted: November 30, 2006

171 13752 Biochemistry 2007, 46, 13752-13761

Enhancing the “A-Rule” of Translesion DNA Synthesis: Promutagenic DNA Synthesis Using Modified Nucleoside Triphosphates† Babho Devadoss,‡ Irene Lee,‡ and Anthony J. Berdis*,§ Departments of Chemistry and Pharmacology, Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106 ReceiVed July 5, 2007; ReVised Manuscript ReceiVed September 6, 2007

ABSTRACT: Abasic sites are mutagenic DNA lesions formed as a consequence of inappropriate modifications to the functional groups present on purines and pyrimidines. In this paper we quantify the ability of the high-fidelity bacteriophage T4 DNA polymerase to incorporate various promutagenic alkylated nucleotides opposite and beyond this class of non-instructional DNA lesions. Kinetic analyses reveal that modified nucleotides such as N 6-methyl-dATP and O6-methyl-dGTP are incorporated opposite an abasic site far more effectively than their unmodified counterparts. The enhanced incorporation is caused by a 10-fold increase in kpol values that correlates with an increase in hydrophobicity as well as changes in the tautomeric form of the nucleobase to resemble adenine. These biophysical features lead to enhanced base-stacking properties that also contribute toward their ability to be easily extended when paired opposite the non- instructional DNA lesion. Surprisingly, misincorporation opposite templating DNA is not enhanced by the increased base-stacking properties of most modified purines. The dichotomy in promutagenic DNA synthesis catalyzed by a high-fidelity polymerase indicates that the dynamics for misreplicating a miscoding versus a non-instructional DNA lesion are different. The collective data set is used to propose models accounting for synergistic enhancements in mutagenesis and the potential to develop treatment-related malignancies as a consequence of utilizing DNA-damaging agents as chemotherapeutic agents.

DNA polymerases play an essential role in maintaining in vivo studies demonstrated that dATP is preferentially genomic integrity by faithfully copying the template strand incorporated opposite an abasic site by various DNA of nucleic acid. Most models of polymerization fidelity polymerases (16-20). This kinetic phenomenon is commonly attribute the veracious transmission of genetic information referred to as the “A-rule” of translesion DNA synthesis to the formation of Watson-Crick hydrogen-bonding inter- (reviewed in ref 21). Our mechanistic studies using the high- actions between the incoming nucleotide and the templating fidelity DNA polymerase from bacteriophage T4 (gp43) base (reviewed in ref 1). Unfortunately, the functional groups demonstrated that dATP is incorporated opposite an abasic that provide these energetically favorable interactions are site ∼100-fold more efficiently than other natural nucleotides highly susceptible to modifications by various DNA-damag- (22). This enhancement is caused through a combination of ing agents (reviewed in ref 2). For example, alkylating agents higher binding affinity coupled with an increase in the rate such as methyl methanesulfonate, dimethylnitrosamine, and constant for polymerization of dATP compared to the other N-methyl-N′-nitro-N-nitrosoguanidine have been shown to natural nucleotides (22). preferentially react with the N3- and N7-positions of adenine Most reports evaluating the effects of various DNA- - in addition to the O6-position of guanine (3 5). These damaging agents have focused on studying promutagenic inappropriate modifications can alter the hydrogen-bonding DNA synthesis caused by misreplication of the modified potential of a templating base to cause mutagenesis (6, 7) templating strand. However, the impact of these alkylating - and induce carcinogenesis (8 10). agents is not solely confined to DNA as nucleoside triph- In addition to the aforementioned modifications, alkylating osphates can also be modified to cause alterations in the agents can also create abasic sites that can arise from the functional groups responsible for hydrogen bonding (23, 24). hydrolysis of a N-glycosidic bond that occurs either Modifications to DNA and nucleotide pools could act in nonenzymatically (11, 12) or via the action of various DNA concert to synergistically decrease polymerization fidelity glycosylases (13-15). The noninstructional nature of an and cause a substantially higher frequency of mutagenesis. abasic site predicts that it would act primarily as a strong Although a wide variety of repair pathways function to avert block for DNA synthesis. However, numerous in vitro and such a catastrophe, their efficiency can be compromised if the cytotoxic insult overwhelms their collective activities. † This research was supported through funding from the National Indeed, this can be a significant complication in the treatment Institutes of Health (CA118408) and the Skin Cancer Foundation to of various cancers as alkylating agents are widely used as AJB. chemotherapeutic modalities against these malignancies (25). * To whom correspondence should be addressed. Phone: (216) 368- In chemotherapy, these agents damage the DNA of cancer 4723. Fax: (216) 368-3395. E-mail: [email protected]. ‡ Department of Chemistry. cells that are rapidly replicating and dividing. Unfortunately, § Department of Pharmacology. this destruction also occurs in other rapidly replicating cells 10.1021/bi701328h CCC: $37.00 © 2007 American Chemical Society Published on Web 11/06/2007 172 Incorporation of Modified Nucleotides Opposite an Abasic Site Biochemistry, Vol. 46, No. 48, 2007 13753 such as bone marrow and gastrointestinal epithelia, which ATP and T4 polynucleotide kinase (GibcoBRL). The assay accounts for side effects including immunosuppression, buffer used in all kinetic studies consisted of 25 mM Tris- nausea, and vomiting. A potentially more devastating OAc (pH 7.5), 150 mM KOAc, and 10 mM 2-mercaptoet- complication is the risk of developing of a secondary cancer hanol. All polymerization reactions were performed at caused by the cytotoxic effects of these DNA-damaging 25 °C and were monitored by analysis of the products on agents (26). 20% sequencing gels as described (30). Gel images were In this paper we evaluate the hypothesis that inappropriate obtained with a Packard PhosphorImager using the Opti- modifications to the templating nucleobase and incoming Quant software supplied by the manufacturer. Product nucleotide can lead to a synergistic decrease in polymerase formation was quantified by measuring the ratio of 32P- fidelity. This was achieved by quantifying the ability of the labeled extended and nonextended primer. The ratios of high-fidelity bacteriophage T4 DNA polymerase to incor- product formation are corrected for substrate in the absence porate and extend beyond an abasic site in the presence of of polymerase (zero point). Corrected ratios are then various alkylated and modified purine analogues. These multiplied by the concentration of primer/template used in kinetic studies reveal that simple alkylation of dATP and each assay to yield the total product. All concentrations are dGTP significantly enhances promutagenic DNA synthesis listed as final solution concentrations. by facilitating incorporation opposite an abasic site. The Determination of the Kinetic Rate and Dissociation resulting structure-activity relationship provides a consistent Constants for dXTP Incorporation. The kinetic parameters, theme in which the catalytic efficiency for incorporation kpol and Kd, for each dXTP were obtained by monitoring the opposite the non-instructional DNA lesion is influenced by rate of product formation using single-turnover reaction the hydrophobic and aromatic properties of the incoming conditions. In these experiments, fixed amounts of gp43 exo- nucleotide. In addition, extension beyond the formed mispair (1 µM) and DNA substrate (250 nM) were used while the is also facilitated by their enhanced base-stacking capabilities. concentration of dXTP was varied from 10 to 500 µM. Although many of these analogues are effectively incorpo- Aliquots of the reaction were quenched into 200 mM EDTA, rated opposite an abasic site, they do not show enhanced pH 7.4, at times ranging from 5 to 240 s. In some instances, misincorporation opposite templating DNA. Collectively, time courses were generated using a rapid quench instrument these data indicate that the mechanism and biophysical forces as previously described (22). These experiments were used to misreplicate templating or non-instructional DNA likewise performed using single-turnover conditions in which lesions are inherently different. 1 µM gp43 exo- and 250 nM DNA substrate were mixed against various concentrations of dNTP (10-500 µM) at time MATERIALS AND METHODS intervals ranging from 0.005 to 10 s. The reactions were quenched through the addition of 350 mM EDTA. Quenched Materials. [γ-32P]ATP was purchased from MP Biomedi- samples were diluted 1:1 with sequencing gel load buffer, cal (Irvine, CA). Unlabeled dNTPs (ultrapure) were obtained and the products were analyzed for product formation by from Pharmacia. MgCl and Trizma base were from Sigma. 2 denaturing gel electrophoresis. Data obtained for single- Urea, acrylamide, and bisacrylamide were from Aldrich. turnover rates in DNA polymerization were fit to eq 1, where Oligonucleotides, including those containing a tetrahydro- furan moiety mimicking an abasic site, were synthesized by - y ) A(1 - e kobsdt) + C (1) Operon Technologies (Alameda, CA). 2-APTP,1 dITP, 8-oxo- dATP, 6-Cl-PTP, 6-Cl-2-APTP, 7-deaza-dATP, N 2-methyl- A is the burst amplitude,kobsd is the first-order rate constant, 6 6 dGTP, O -methyl-dGTP, and N -methyl-dATP were obtained t is time, and C is a defined constant. Data for the dependency from TriLink BioTechnologies (San Diego, CA), while of kobsd versus dXTP concentration were fit to the equation 7-deaza-dGTP was obtained from Sigma in greater than 99% describing a rectangular hyperbola (eq 2), where kobsd is the purity. All other materials were obtained from commercial sources and were of the highest available quality. The ) + kobsd kpol[dXTP]/Kd [dXTP] (2) exonuclease-deficient mutant of gp43 (Asp-219 to Ala mutation) was purified and quantified as previously described apparent first-order rate constant, kpol is the maximal po- (27, 28). lymerization rate constant, Kd is the kinetic dissociation General Methods. Single-stranded and duplex DNA were constant for dXTP, and [dXTP] is the concentration of purified and quantified as previously described (29). 5′-Ends nucleotide substrate. of the primer and template strands were labeled using [γ-32P]- Extension Beyond an Abasic Site. Single-turnover condi- tions were used to measure the rates of extension beyond a - 1 Abbreviations: TBE, Tris-HCl/borate/EDTA; EDTA, ethylene- dXMP:abasic site mispair. gp43 exo (1 µM) was incubated diaminetetraacetate, sodium salt; dNTP, deoxynucleoside triphosphate; with 500 nM DNA (13/20SP-mer) in assay buffer containing dXTP, unnatural deoxynucleoside triphosphate; 2-APTP, 2-amino-2′- ′ ′ ′ EDTA (100 µM) and mixed with 100 µM dXTP and 10 mM deoxyadenosine 5 -triphosphate; dITP, 2 -deoxyinosine 5 -triphosphate; ∼ 6-Cl-PTP, 6-chloropurine-2′-deoxyadenosine 5′-triphosphate; 6-Cl-2- magnesium acetate. After 60 s, 900 µM dGTP (the correct APTP, 6-chloro-2-amino-2′-deoxyriboside 5′-triphosphate; 7-deaza- dNTP for the next three positions) was added. Aliquots of dATP, 7-deaza-2′-deoxyadenosine 5′-triphosphate; 7-deaza-dGTP, 7-dea- the reactions were quenched with 500 mM EDTA at variable za-2′-deoxyguanosine 5′-triphosphate; N 2-methyl-dGTP, N 2-methyl- - 2′-deoxyguanosine 5′-triphosphate; O6-methyl-dGTP, O6-methyl-2′- times (5 900 s) and analyzed as described above. deoxyguanosine 5′-triphosphate; N 6-methyl-dATP, N 6-methyl-2′- deoxyadenosine 5′-triphosphate; 5-NITP, 5-nitroindolyl-2′-deoxyriboside RESULTS AND DISCUSSION triphosphate; 5-PhITP, 5-phenylindolyl-2′-deoxyriboside triphosphate; gp43 exo-, an exonuclease-deficient mutant of the bacteriophage T4 Kinetic Parameters for Incorporation of Alkylated Purines DNA polymerase. Opposite an Abasic Site. The focus of this study is to 173 13754 Biochemistry, Vol. 46, No. 48, 2007 Devadoss et al.

FIGURE 1: (A) Structures of 2′-deoxynucleoside triphosphates used or referred to in this study, i.e., dATP, dGTP, N 6-methyl-dATP, 6-Cl- PTP, dITP, O6-methyl-dGTP, N 2-methyl-dGTP, 2-APTP, 6-Cl-2-APTP, 7-deaza-dATP, 7-deaza-dGTP, 5-NITP, and 5-PhITP. For convenience, R is used to represent the deoxyribose triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes any of the four natural nucleobases or the presence of a tetrahydrofuran moiety that functionally mimics an abasic site. understand the molecular mechanism for the misreplication exponential process (eq 1) to obtain kobsd values. The plot of 6 of an abasic site (Figure 1A), a DNA lesion that can kobsd versus N -methyl-dATP concentration is hyperbolic culminate from the alkylation of purines and pyrimidines (Figure 2B) and was fit to the equation describing a (3-5). Upon encountering this non-instructional DNA lesion, rectangular hyperbola to obtain a Kd of 190 ( 45 µM, a kpol -1 -1 -1 most polymerases preferentially incorporate dATP and of 5.6 ( 0.6 s , and a kpol/Kd of 28 420 M s . subsequently extend beyond the formed mispair (16-20)to The 7-fold greater catalytic efficiency for N 6-methyl-dATP propagate a potential genomic error. The question asked in compared to dATP (28 420 M-1 s-1 versus 4300 M-1 s-1, this paper is whether promutagenic DNA synthesis can be respectively) indicates that the modified nucleotide is more exacerbated through modifications to both the DNA template promutagenic than the parental nucleotide. The enhanced and the incoming nucleoside triphosphate. kinetic behavior for N 6-methyl-dATP does not arise from 6 To evaluate this question, we examined whether subtle an increase in binding affinity as the Kd of 190 µM for N - modifications to dATP or dGTP can influence the dynamics methyl-dATP is ∼5-fold higher than the Kd of 35 µM for 6 -1 6 of misincorporation. The first analogue tested is N -methyl- dATP (22). Instead, the kpol value of 5.6 s for N -methyl- dATP,2 a modified purine that can arise through simple dATP is ∼40-fold faster than that for dATP (0.15 s-1 (22)). alkylation at the N6-position of adenine (Figure 1B). Representative data provided in Figure 2A illustrate the 2 Alkylating agents predominantly modify dATP at the heterocyclic dependency on the rate constant in primer elongation as a nitrogens (N3 and N7) as well as at the exocyclic amino group (N6- 6 position) (6). Although N3-alkylated dATP is well characterized, the function of N -methyl-dATP concentration. Each time course formation and physiological consequences of N 6-methyl-dATP are in primer elongation was fit to the equation defining a single- poorly understood. 174 Incorporation of Modified Nucleotides Opposite an Abasic Site Biochemistry, Vol. 46, No. 48, 2007 13755 increases the potential for promutagenic synthesis opposite an abasic site. The enhanced catalytic efficiency is again caused by an increased kpol value rather than an influence on binding affinity. At face value, the increased kpol values coincide with changes associated with solvation energies between modified and unmodified nucleobases. However, -1 this explanation is incomplete since the kpol of 0.11 s measured with N 2-methyl-dGTP is ∼9-fold slower than that of 0.98 s-1 for O6-methyl-dGTP. This difference corresponds to a relative change in Gibb’s free energy (∆∆G) of 1.3 kcal/ mol and is significantly less that the 2.3 kcal/mol difference in solvation energies between the two analogues (Table 1). Similar observations exist comparing alkylated nucleotides with their unmodified counterparts as the change in ∆∆G associated with solvation energies of the nucleotides does not equal the energetic differences associated with increases in the kpol values. This analysis clearly indicates that other biophysical parameters must also contribute to account for the energetic differences. We propose that differences in the aromaticity of the nucleotides as manifest in the different tautomeric forms of N 2-methyl-dGTP versus O6-methyl- dGTP influence their incorporation opposite the non-instruc- tional lesion. This prediction is reasonable since changes in tautomeric form are known to influence the base-stacking capacity of a nucleotide (31, 32). This hypothesis was tested by measuring the kinetic FIGURE 2: (A) Dependency of N 6-methyl-dATP concentration on parameters for dITP (Figure 1B), a nucleotide formed via the observed rate constant in primer elongation as measured using the deamination of dATP. If hydrophobicity alone dictates single-turnover conditions. The following concentrations of N 6- b 0 ] incorporation efficiency opposite an abasic site, then the methyl-dATP were used: 25 µM( ), 50 µM( ), 100 µM( ), ∼ 250 µM(+), and 500 µM(4). The solid lines represent the fit of resulting 5 kcal/mol decrease in the solvation energy each time course to a single-exponential process. (B) The observed associated with this modification should enhance the incor- rate constants for incorporation (b) were plotted against N 6-methyl- poration of dITP opposite the lesion. Contrary to this dATP concentration and fit to the equation defining a rectangular prediction, we find that dITP is incorporated very poorly hyperbola to determine values corresponding to Kd, kpol, and -1 -1 k /K . opposite an abasic site with a kpol/Kd value of 430 M s . pol d -1 In fact, the low kpol of 0.044 s and relatively high Kd of ) The dramatic enhancement in the k value suggests that 103 µM are nearly identical to those for dGTP (kpol 0.023 pol -1 ) increasing the hydrophobicity and/or overall size of the s and Kd 130 µM(22)). Indeed, the striking similarity nucleotide facilitates the conformational change step that in the kinetic behavior of dITP and dGTP coincides with precedes phosphoryl transfer. the similarities in tautomeric form. We next evaluated whether alkylation of other natural The collective data set indicates that the aromatic nature nucleotides also increases their promutagenic potential. The of the nucleotide in addition to its hydrophobicity plays a incorporation of O6-methyl-dGTP and N 2-methyl-dGTP significant role during translesion DNA synthesis to enhance opposite an abasic site was tested since alkylation at either the promutagenic replication of an abasic site. At the position leads to an overall increase in size and hydrophobic- molecular level, this enhancement is achieved through ity compared to those of dGTP (refer to Table 2). The increases in the kpol value that reflects the enhanced base- catalytic efficiency for O6-methyl-dGTP is ∼30-fold greater stacking capabilities of the nucleotide. On the other hand, compared to that of the incorporation of dGTP (compare binding affinity appears to be adversely affected by any 5400 M-1 s-1 with 180 M-1 s-1, respectively).3 Alkylation increase in the hydrophobic nature of the incoming nucle- at the O6-position of dGTP leads to an effect similar to that otide. As discussed below, one possibility is that the observed with N 6-methyl-dATP, i.e., a significant increase functional groups on dATP that provide hydrogen-bonding in the k value concomitant with decreased binding affinity. interactions are in fact essential for achieving optimal binding pol in the absence of templating information. In fact, it is striking that the 40-fold faster kpol value with O6-methyl-dGTP versus dGTP (compare 0.98 s-1 with 0.023 Kinetic Parameters for the Incorporation of Modified -1 Purines Opposite an Abasic Site. We next tested the ability s , respectively) is identical to the 40-fold enhancement in - 6 of gp43 exo to incorporate various unnatural purine kpol values between N -methyl-dATP and dATP. The ∼3-fold higher catalytic efficiency for N 2-methyl- triphosphates such as 7-deaza-dATP, 6-Cl-PTP, and 6-Cl- dGTP compared to dGTP also indicates that alkylation 2-APTP (Figure 1B) opposite an abasic site. The various atomic substitutions and permutations of functional groups allow us to further evaluate the role of hydrophobicity and 3 6 The magnitude of this effect for O -methyl-dGTP is greater than aromatic interactions during translesion DNA synthesis. K that for N 6-methyl-dATP. However, the larger magnitude for O6-methyl- d dGTP reflects the poor kinetics by which dGTP is incorporated opposite and kpol values for this series of purine analogue were the abasic site. measured as described above and are summarized in Table 175 13756 Biochemistry, Vol. 46, No. 48, 2007 Devadoss et al.

FIGURE 3: (A) Experimental paradigm used to measure insertion and extension beyond an abasic site lesion. A preincubated solution of 1 µM gp43 exo- and 500 nM 5′-labeled 13/20SP-mer was mixed with 50 µM dXTP to initiate the reaction. After 2 min, an aliquot of the reaction was quenched with 200 mM EDTA (denoted as “Inc”) to measure insertion opposite the lesion. dGTP (900 µM) was then added, and aliquots of the reaction were quenched with 200 mM EDTA at 60 s. (B) Denaturing gel electrophoresis reveals that gp43 exo- extends beyond dATP, N 6-methyl-dATP, 6-Cl-PTP, O6-methyl-dGTP, 6-Cl-2-APTP, and N 7-deaza-dATP when paired opposite an abasic site.

Table 1: Summary of Kinetic Parameters for the Incorporation of Modified Nucleotides Opposite an Abasic Sitea surface dipole solvation b b b KD kpol kpol/KD area moment energy tautomeric analogue (µM) (s-1) (M-1 s-1) (Å2) (D) (kcal/mol) formc dATPd 35 ( 5 0.15 ( 0.01 4 300 143.0 2.38 -19.258 dATP dGTPd 130 ( 5 0.023 ( 0.005 180 152.5 7.18 -26.009 dGTP N 6-methyl-dATP 190 ( 45 5.6 ( 0.6 29 500 165.0 2.16 -16.322 dATP O6-methyl-dGTP 181 ( 35 0.98 ( 0.08 5 400 174.5 2.94 -19.998 dATP N 2-methyl-dGTP 245 ( 68 0.12 ( 0.02 490 173.1 7.54 -22.306 dGTP dITP 103 ( 14 0.044 ( 0.003 430 139.0 5.63 -21.790 dGTP 7-deaza-dATP 197 ( 40 1.4 ( 0.1 7 100 148.7 3.64 -17.846 dATP 7-deaza-dGTP 215 ( 91 0.11 ( 0.02 500 158.2 4.95 -24.247 dGTP 6-Cl-PTP 83 ( 16 0.28 ( 0.02 3 380 145.2 4.99 -16.449 dATP 6-Cl-2-APTP 71 ( 20 0.12 ( 0.01 1 700 158.7 4.83 -19.063 dATP 2-APTP 180 ( 23 0.23 ( 0.02 1 300 143.2 3.10 -19.142 dATP 5-NITPe 18 ( 3 126 ( 7 7 000 000 171.4 7.81 -7.381 NAg 5-PhITPf 14 ( 353( 4 3 800 000 223.2 3.31 -5.532 NA a Assays were performed using 1 µM gp43 exo-, 250 nM 13-20SP-mer, and variable concentrations of unnatural nucleotide in the presence of 10 mM Mg2+. b Surface areas (used as an indicator of the relative size of the nucleobase), dipole moments (D), and solvation energies for each nucleobase were calculated using Spartan ’04 software. c The tautomeric form refers to whether the purine analogue has the same tautomeric form as dATP or dGTP. d Values taken from ref 6. e Values taken from ref 47. f Values taken from ref 48. g NA ) not applicable

1. In general, modifications and/or atomic substitutions that 0.12 s-1 measured for 6-Cl-2-APTP is 6-fold faster than that increase the hydrophobicity of a nucleotide generally increase of 0.023 s-1 reported for dGTP (22). However, this increase the rate constant for incorporation while reducing binding is most likely caused by a combination of a change in affinity opposite an abasic site. 7-Deaza-dATP provides a hydrophobicity and a change in tautomeric form to resemble clear example of this kinetic phenomenon as the kpol value dATP, the preferred natural nucleotide. of 1.5 s-1 is 10-fold faster than that of 0.15 s-1 for dATP Another unique feature is that the binding affinities for (22) while the Kd value of 200 µMis∼6-fold higher than 6-Cl-PTP and 6-Cl-2-APTP are among the lowest measured the Kd of 35 µM for dATP. Similar observations are obtained for all the modified nucleotides tested in this study. As argued when the kinetic parameters for 7-deaza-dGTP are compared above, this result suggests that the 6-position of a purine with those for dGTP. may play an important role in the binding of natural and The replacement of a hydrogen-bonding group with a non- unnatural nucleotides during translesion DNA synthesis. This hydrogen-bonding group does not substantially enhance the argument is supported by the high Kd value of 180 µM for dynamics of translesion DNA synthesis. For example, 6-Cl- 2-APTP, a constitutional analogue of dATP in which the PTP shows only a modest 2-fold increase in kpol despite a N6 exocyclic amine is permutated to the 2-position (Figure 2.81 kcal/mol reduction in solvation energy. The same 1B). Although this permutation has a strong effect on binding modification of dGTP yields a larger effect as the kpol of affinity, the influence on kpol is minimal since the value of 176 Incorporation of Modified Nucleotides Opposite an Abasic Site Biochemistry, Vol. 46, No. 48, 2007 13757

Table 2: Summary of Kinetic Rate Constants for Extension beyond an Abasic Site Catalyzed by gp43 exo- a solvation solvation kext energy kext energy dXTP (s-1) (kcal/mol) dXTP (s-1) (kcal/mol) “dATP-like” Analogues dATP 0.25 ( 0.01 -19.258 6-Cl-2-APTP 0.033 ( 0.004 -19.063 7-deaza-dATP 0.32 ( 0.03 -17.846 6-Cl-PTP 0.031 ( 0.003 -16.449 N 6-methyl-dATP 0.71 ( 0.04 -16.322 2-APTP NDb -19.142 O6-methyl-dGTP 0.092 ( 0.009 -22.306 “dGTP-like” Analogues dGTP 0.005 ( 0.001 -26.009 dITP ND -21.790 N 2-methyl-dGTP ND -19.998 7-deaza-dGTP ND -24.247 a Insertion and extension beyond an abasic site lesion were measured by preincubating gp43 exo- (1 µM) with 5′-labeled 13/20SP-mer (500 nM) and then mixing with a 50 µM concentration of modified nucleotide to initiate the reaction. After 2 min, an aliquot of the reaction was quenched with 200 mM EDTA (denoted as “Inc”) to measure insertion opposite the lesion. dGTP (900 µM) was then added, and aliquots of the reaction were quenched with 200 mM EDTA at time intervals raging from 5 to 300 s. The generated time courses were fit to eq 1 to define kext, the rate constant for extension beyond the formed mispair. b ND ) not detected.

Table 3: Summary of Kinetic Parameters for the Incorporation of Unnatural Nucleotides Opposite T and Ca thymine cytosine

KD kpol kpol/KD KD kpol kpol/KD analogue (µM) (s-1) (M-1 s-1) (µM) (s-1) (M-1 s-1) dATPb 10 ( 0.5 100 ( 10 10 000 000 ND ND <200c dGTP 500 ( 200 0.04 ( 0.01 75 5 ( 247( 4 9 400 000 N 6-methyl-dATP 22 ( 13 82 ( 13 4 000 000 ND ND <40c O6-methyl-dGTP 465 ( 190 115 ( 27 247 310 116 ( 34 20.3 ( 2 175 000 N 2-methyl-dGTP 466 ( 177 0.07 ( 0.01 150 60 ( 35 1.7 ( 0.4 28 330 dITP 70 ( 19 0.080 ( 0.006 1 140 7 ( 549( 10 7 000 000 7-deaza-dATP 7 ( 280( 9 11 430 000 270 ( 90 9 ( 2 33 300 7-deaza-dGTP 850 ( 400 0.14 ( 0.04 164 2.4 ( 0.7 57 ( 4 23 750 000 2-APTP 25 ( 11 119 ( 20 4 760 000 90 ( 38 1.6 ( 0.2 18 100 6-Cl-PTP 25 ( 513( 1 520 000 395 ( 85 0.040 ( 0.003 105 a Assays were performed using 1 µM gp43 exo-, 250 nM 13/20T-mer or 13/20C-mer, and variable concentrations of natural or modified nucleotide in the presence of 10 mM Mg2+. b Values taken from ref 29. c Estimates were calculated from the linear portion of the Michaelis-Menten plot.

0.23 s-1 is nearly identical, within error, to that of 0.15 s-1 predicted favorable base-stacking interactions opposite an for dATP. The similarity in tautomeric form and solvation abasic site. energies between 2-APTP and dATP again correlates with Again, it is important to emphasize that the increase in the identical kpol values. base-stacking capacity depends upon the hydrophobic and Modified Purines Enhance Elongation beyond an Abasic aromatic nature of the nucleotide. This is apparent by Site. gp43 exo- extends beyond an abasic site only when examining the ability of gp43 exo- to elongate beyond N 2- either dAMP or dGMP is placed opposite the lesion (22, methyl-dGTP. Although alkylation at the N2-position of 33). The dynamics of this process are proposed to reflect dGTP increases its hydrophobicity, this does not lead to an the positioning of either purine in an interhelical position enhancement in primer elongation. Furthermore, the lack of that likely reflects their enhanced base-stacking capabilities extension beyond dITP or N 7-deaza-dGTP is arguably caused compared to those of pyrimidines (34, 35). This model leads by their weaker base-stacking capabilities that reflect reduc- to a testable prediction: modified purines that possess tions in their aromatic nature. Finally, the influence of enhanced base-stacking capabilities should be elongated aromaticity is also evident in the ∼20-fold increase in kext more easily than their unmodified counterparts. The biologi- for O6-methyl-dGTP that results in a change in tautomeric cal ramifications of this model are obvious since the ability form caused by alkylation at the O6-position. of a modified nucleotide to be elongated predicts a higher The lone exception to this proposed model is 2-APTP. promutagenic potential. This model was tested using the Despite being similar to dATP with respect to tautomeric experimental protocol outlined in Figure 3A that monitors form and hydrophobicity, this analogue is not extended when the ability of gp43 exo- to extend beyond the various paired opposite an abasic site. Although the molecular reason modified purines. Gel electrophoresis data provided in Figure for this phenomenon is currently unknown, one possibility 3B reveal that most hydrophobic nucleotides that resemble is that removal of a functional group at the C6-position dATP are easily elongated. In contrast, analogues that are prevents contacts between the minor groove of DNA and more hydrophilic and/or that resemble dGTP are more the DNA polymerase which are important for polymerase refractory to elongation. As summarized in Table 2, the rate translocation (36, 37). Consistent with this mechanism is the 6 constants for extension (kext) of analogues such as N -methyl- fact that 6-Cl-PTP is elongated ∼8-fold slower compared to dATP and 7-deaza-dATP are ∼1.5-3-fold faster than dATP. These latter cases suggest that perturbations to the that of dATP. This enhancement coincides with their exocyclic amino group may influence the kinetics of elonga- 177 13758 Biochemistry, Vol. 46, No. 48, 2007 Devadoss et al.

templating base or through diminution in incorporation opposite its predicted “correct” pairing partner or both. Distinguishing among these possibilities is important to accurately interpret how DNA synthesis is perturbed as a consequence of the nucleotide modifications. Using this approach, we find that both unmodified nucleotides dATP and dGTP are very selective since their calculated SF values are >104. These high SF values are intuitively obvious since each nucleotide is predicted to exclusively interact with its complementary partner due to a combination of hydrogen-bonding interactions and steric constraints. Furthermore, these features are proposed to hinder their misincorporation opposite their noncognate partners. In this regard, it is surprising that certain analogues such as 7-deaza-dATP and N 2-methyl-dGTP, which have minimal perturbations toward steric fit and hydrogen-bonding interactions, have significantly lower SF values compared to their unmodified counterparts. This is interesting since both analogues are also incorporated opposite an abasic site with a relatively high efficiency. At face value, these coincidences suggest that simply increasing the hydrophobic- ity of a nucleotide will cause a unilateral decrease in fidelity. Indeed, this could describe the behavior of 7-deaza-dATP as the low SF value of 340 is caused by a significant increase in the misincorporation opposite cytidine while correct incorporation opposite thymine is left unperturbed. However, a universal “hydrophobic effect” is unlikely since the low FIGURE 4: (A) Representation of the SF for each modified SF value of 190 for N 2-methyl-dGTP is not caused by an ) nucleotide for incorporation opposite thymine or cytidine. SF increase for misinsertion opposite thymine. Instead, the (kpol/Kd)comp/(kpol/Kd)noncomp. Modified nucleotides are classified as those possessing high SF values of >103, those with low SF values reduced fidelity is caused by a surprisingly large decrease 3 of <10 , and those with no selectivity (SF values of ∼1). (B) kpol/ in incorporation opposite cytidine, its predicted complemen- Kd values for each nucleotide as a function of templating nucleobase. tary partner. Red bars represent incorporation opposite thymine, while black bars The best example arguing against a hydrophobic effect represent incorporation opposite cytosine. causing reduced fidelity in the presence of templating information is provided in the kinetic data for N 6-methyl- tion. However, the collective data set demonstrates that the dATP. On one hand, this highly promutagenic nucleotide base-stacking properties of an incoming nucleotide increase displays the highest catalytic efficiency for incorporation its promutagenic potential by directly influencing incorpora- opposite an abasic site. However, it is remarkable that N 6- tion and extension beyond an abasic site. methyl-dATP maintains exquisite selectivity for incorporation Influence of Hydrophobicity and Tautomeric Form on opposite thymine versus cytidine. In fact, the SF value for Misincorporation Opposite Templating Bases. We next N 6-methyl-dATP is actually 2-fold higher than that for dATP questioned whether the enhanced base-stacking capacity of (compare 114 300 versus 50 000, respectively). This feature these modified nucleotides would also contribute toward is not unique to N 6-methyl-dATP as similar trends are also facilitating misincorporation opposite templating nucleobases. observed with other hydrophobic analogues such as 7-deaza- This was initially tested by measuring Kd, kpol, and kpol/Kd dGTP and 6-Cl-dATP which maintain high SF values of values for this series of purine analogues opposite thymine >103. or cytidine, which are predicted to be their complementary One nucleotide that bears special emphasis is O6-methyl- partners (summarized in Table 3). Figure 4A provides a dGTP as this nucleotide owns the distinction of being the graphical representation of the selectivity factor for each most promutagenic nucleotide identified in this study. With modified nucleotide for incorporation opposite thymine or respect to translesion DNA synthesis, this nucleotide is cytidine. The selectivity factor (SF) is defined as the ratio effectively incorporated opposite an abasic site with a -1 -1 of kpol/Kd for nucleotide incorporation opposite its predicted relatively high catalytic efficiency of 5400 M s (Table complementary partner versus its noncomplementary partner 1). In addition, O6-methyl-dGTP is incorporated opposite (SF ) (kpol/Kd)comp/(kpol/Kd)noncomp). This analysis allows us thymine and cytidine with a low SF value of ∼1, which to classify the modified nucleotides into three distinct indicates complete promiscuity for incorporation opposite categories, those with high SF values of >103, those with pyrimidines. It is quite surprising that the similar catalytic low SF values of <103, and those with no selectivity (SF efficiencies of 247 310 and 175 000 M-1 s-1 for incorpora- values of ∼1). In addition, we provide a plot of the kpol/Kd tion opposite thymine and cytidine, respectively, occur values for each nucleotide as a function of templating through differential perturbations in the measured Kd and -1 nucleobase (Figure 4B). This latter analysis is important since kpol values. On one hand, the fast kpol of 116 s for it indicates whether the SF values for various nucleotides incorporation opposite thymine likely reflects the fact that are caused by an enhancement in the misreplication of a O6-methyl-dGTP has the same tautomeric form as dATP. 178 Incorporation of Modified Nucleotides Opposite an Abasic Site Biochemistry, Vol. 46, No. 48, 2007 13759

This interpretation would support the fast kpol value of ∼1 anism by which certain nucleoside analogues such as s-1 measured for the incorporation of O6-methyl-dGTP ribavirin exert their antiviral effects. opposite an abasic site. However, the binding affinity of O6- methyl-dGTP opposite thymine is dramatically reduced as CONCLUSIONS manifest in the high K value of 465 µM. This suggests that d In this paper we demonstrate that alkylated purine triph- ground-state binding opposite a templating base is influenced osphates can significantly enhance the promutagenic proper- predominantly by hydrogen-bonding and/or steric-fit con- ties of an abasic site. The provided kinetic data yield further straints rather than by base-stacking interactions. insights into the molecular mechanism of translesion syn- We performed additional experiments measuring the thesis. During replication of an abasic site, we argue that incorporation of all modified purine triphosphates opposite the enzymatic conformational change step (as reflected by noncognate partners, adenine and guanine (gel electrophoretic the k value) is influenced by the hydrophobic and aromatic data and a summary of rate constants are provided as pol nature of the incoming nucleotide.4 This conclusion is Supporting Information Figure 1). All of the modified purines consistent with our published model derived from character- are poorly incorporated opposite adenine or guanine as they izing the incorporation of various 5-substituted indolyl possess estimated catalytic efficiencies of less than 10 M-1 nucleoside triphosphates opposite this non-instructional DNA s-1. In fact, the rate constant for the incorporation of nearly lesion (46). Non-natural nucleotides such as 5-NITP and all analogues is <0.01 s-1 even at nucleotide concentrations 5-PhITP (Figure 1B) are incorporated opposite the abasic greater than 300 µM. In this regard, it is surprising that dATP site with incredibly high catalytic efficiencies (>106 M-1 is incorporated opposite adenine with a relatively high s-1)(47, 48). Both analogues have low affinity constants catalytic efficiency of 420 M-1 s-1 (Supporting Information (K values <20 µM) coupled with fast incorporation rate Figure 2A). A similar phenomenon is observed for the d constants (k values >50 s-1). The faster k values incorporation of dGTP opposite guanine in which the overall pol pol measured with these 5-substituted indolyl nucleotides likely catalytic efficiency is estimated to be ∼100 M-1 s-1 reflects their enhanced base-stacking capabilities that origi- (Supporting Information Figure 2B). nate from their large π-electron surface area and hydrophobic Collectively, the kinetic data obtained using these modified nature. purine analogues do not provide evidence for a strong correlation between their hydrophobic and/or aromatic nature The ability of various modified purines to be efficiently and the ability to form a mispair. In this regard, the dynamics elongated has important biological ramifications, especially by which a polymerase misreplicates a templating base within the context of replicating non-instructional DNA appear to be vastly different from the dynamics for the lesions such as an abasic site. Simple alkylation of dATP or misincorporation of a nucleotide opposite an abasic site. This dGTP enhances the efficiency of translesion DNA synthesis conclusion has several important ramifications. First, differ- that could cause a synergistic increase in mutagenesis under ences in the underlying mechanism by which damaged DNA certain circumstances. The provocative implication of this is misreplicated provide insight as to why the mutagenic conclusion is the potential risk of certain chemotherapeutic spectra of closely related DNA-damaging agents are different. modalities to induce mutagenesis that may result in an For example, dCTP and dTTP are typically incorporated additional prooncogenic event. Indeed, the administration of opposite damaged nucleobases such as O6-methylguanine and certain cytotoxic DNA-damaging agents such as temozolo- O6-ethylguanine that contain small modifications (38, 39). mide and cyclophosphamide is associated with a 10-fold This implies that these lesions are replicated using features higher risk of developing a secondary cancer (49). associated with classical hydrogen-bonding and steric-fit Finally, the dynamics by which these modified purines constraints much the same as for an unmodified guanine. are misincorporated are highly dependent upon the presence However, the introduction of larger substituent groups at the or absence of templating information. On one hand, increas- O6-position of guanine gives rises to a different mutational ing the hydrophobicity and aromaticity of a nucleotide leads spectrum in which dATP or dGTP is preferentially incor- to enhanced efficiency in incorporation and extension beyond porated (40, 41). In this latter case, the preferential incor- an abasic site. However, a much more complex scenario poration of dATP argues that larger DNA lesions are exists during misinsertion opposite templating bases. In replicated via a “transient abasic site intermediate”. In any general, the catalytic efficiency for misinsertion depends event, this information could be used to develop a predictive more upon alterations in hydrogen-bonding interactions and index for the mutagenic potential of other DNA-damaging shape complementarity as opposed to perturbations in agents. Another practical application is toward the rational hydrophobicity. Of course, unambiguous conclusions are design of antiviral agents that take advantage of low-fidelity difficult to make since modifying the functional group of replication catalyzed by most viral polymerases. Lower an incoming nucleotide influences all three biophysical replication fidelity is generally tolerated by many viruses features to varying degrees. This last feature is important as (42). However, the introduction of too many mutations, it questions whether a truly universal mechanism of polym- referred to as error catastrophe, may cause a loss of viral erization exists during the replication of normal versus fitness and viability (43). This phenomenon has led to the damaged DNA. The data presented here suggest that different development of a chemotherapeutic strategy termed “lethal mutagenesis” in which promutagenic nucleotides are used 4 We have previously demonstrated that the incorporation of dATP to increase the mutation frequency of viruses beyond viability and other unnatural nucleotides such as 5-NITP and 5-PhITP opposite to induce error catastrophe (44, 45). While lethal mutagenesis an abasic site is limited by the conformational change step preceding phosphoryl transfer (22, 33, 46, 48). 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38. Spratt, T. E., and Levy, D. E. (1997) Structure of the hydrogen 44. Loeb, L. A., Essigmann, J. M., Kazazi, F., Zhang, J., Rose, K. bonding complex of O6-methylguanine with cytosine and thymine D., and Mullins, J. I. (1999) Lethal mutagenesis of HIV with during DNA replication, Nucleic Acids Res. 25, 3354-3361. mutagenic nucleoside analogs, Proc. Natl. Acad. Sci. U.S.A. 96, 39. Perrino, F. W., Blans, P., Harvey, S., Gelhaus, S. L., McGrath, 1492-1497. C., Akman, S. A., Jenkins, G. S., LaCourse, W. R., and Fishbein, 45. Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, J. C. (2003) The N2-ethylguanine and the O6-ethyl- and O6- Z., Andino, R., and Cameron, C. E. (2000) The broad-spectrum methylguanine lesions in DNA: contrasting responses from the antiviral ribonucleoside ribavirin is an RNA virus mutagen, Nat. “bypass” DNA polymerase eta and the replicative DNA poly- Med. 6, 1375-1379. merase alpha, Chem. Res. Toxicol. 16, 1616-1623. 46. Devadoss, B., Lee, I., and Berdis, A. J. (2007) Is a thymine dimer 40. Perlow, R. A., and Broyde, S. (2002) Toward understanding the replicated via a transient abasic site intermediate? A comparative mutagenicity of an environmental carcinogen: Structural insights study using non-natural nucleotides, Biochemistry 46, 4486-4498. into nucleotide incorporation preferences, J. Mol. Biol. 322 (2), 47. Reineks, E. Z., and Berdis, A. J. (2004) Evaluating the contribution 291-309. of base stacking during translesion DNA replication, Biochemistry 41. Nivard, J. M. M., Pastink, A., and Vogel, W. E. (1992) Molecular 43, 393-404. V analysis of mutations induced in the ermilion gene of Drosophila 48. Zhang, X., Lee, I., and Berdis, A. J. (2005) The use of nonnatural - melanogaster by methyl methanesulfonate, Genetics 131, 673 nucleotides to probe the contributions of shape complementarity 682. and pi-electron surface area during DNA polymerization, Bio- 42. Svarovskaia, E. S., Cheslock, S. R., Zhang, W. H., Hu, W. S., chemistry 44, 13101-13110. and Pathak, V. K. (2003) Retroviral mutation rates and reverse - 49. Smith, M. A., McCaffrey, R. P., and Karp, J. E. (1996) The transcriptase fidelity, Front. Biosci. 8, d117 134. secondary leukemias: challenges and research directions, J. Natl. 43. Anderson, J. P., Daifuku, R., and Loeb, L. A. (2004) Viral error Cancer Inst. 88, 407-418. catastrophe by mutagenic nucleosides, Annu. ReV. Microbiol. 58, 183-205. BI701328H

181 4486 Biochemistry 2007, 46, 4486-4498

Is a Thymine Dimer Replicated via a Transient Abasic Site Intermediate? A Comparative Study Using Non-Natural Nucleotides† Babho Devadoss,‡ Irene Lee,‡ and Anthony J. Berdis*,§ Departments of Chemistry and Pharmacology, Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106 ReceiVed NoVember 27, 2006; ReVised Manuscript ReceiVed January 31, 2007

ABSTRACT: UV light causes the formation of thymine dimers that can be misreplicated to induce mutagenesis and carcinogenesis. This report describes the use of a series of non-natural indolyl nucleotides in probing the ability of the high-fidelity bacteriophage T4 DNA polymerase to replicate this class of DNA lesion. Kinetic data reveal that indolyl analogues containing large π-electron surface areas are incorporated opposite the thymine dimer almost as effectively as an abasic site, a noninstructional lesion. However, there are notable differences in the kinetic parameters for each DNA lesion that indicate distinct mechanisms for their replication. For example, the rate constants for incorporation opposite a thymine dimer are considerably slower than those measured opposite an abasic site. In addition, the magnitude of these rate constants depends equally upon contributions from π-electron density and the overall size of the analogue. In contrast, binding of a nucleotide opposite a thymine dimer is directly correlated with the overall π-electron surface area of the incoming dXTP. In addition to defining the kinetics of polymerization, we also provide the first reported characterization of the enzymatic removal of natural and non-natural nucleotides paired opposite a thymine dimer through exonuclease degradation or pyrophosphorolysis activity. Surprisingly, the exonuclease activity of the bacteriophage enzyme is activated by a thymine dimer but not by an abasic site. This dichotomy suggests that the polymerase can “sense” bulky lesions to partition the damaged DNA into the exonuclease domain. The data for both nucleotide incorporation and excision are used to propose models accounting for polymerase “switching” during translesion DNA synthesis.

The ability of DNA polymerases to bypass various DNA the mechanism of nucleotide incorporation opposite and lesions is considered to be a promutagenic event that can beyond a thymine dimer. Although it has been demonstrated lead to carcinogenesis (1). One clear example of the causal that DNA polymerases can incorporate dNTPs opposite UV- link between the misreplication of damaged DNA and induced DNA lesions (7-9), there are few reports providing carcinogenesis is the development of skin cancer (reviewed detailed kinetic analyses of this activity. One exception, in ref 2). The primary causative agent of skin cancer is solar however, is the bacteriophage T7 DNA polymerase that has UV light that catalyzes the formation of dipyrimidine been extensively evaluated through kinetic and structural photoproducts such as cyclobutane pyrimidine dimers and studies (13-15). This high-fidelity DNA polymerase is pyrimidine 6-4 pyrimidone photoproducts (reviewed in ref proposed to replicate a thymine dimer as a noninstructional 3). Although these lesions are repaired by several distinct lesion, i.e., an abasic site, as opposed to a misinstructional DNA repair pathways (4-6), they are also inappropriately lesion (13). This conclusion was based upon comparative replicated by various DNA polymerases (7-9). Indeed, nucleotide selectivity studies demonstrating that the T7 DNA insufficient repair followed by errors in replication produces polymerase preferentially incorporates pyrene triphosphate characteristic mutations in dipyrimidine sequences that (dPTP)1 versus dATP opposite either DNA lesion (13, 14). represent initiating events in cancer (10-12). For example, At the molecular level, this result is intriguing since the squamous cell carcinoma involves mutations in the p53 gene structures of these lesions differ significantly (Figure 1A). (10), while basal cell carcinoma (11) and melanoma (12) Regardless, this model was supported by structural evidence involve mutations in the PATCHED gene and the p16 gene, of the T7 exo- DNA polymerase bound at a thymine dimer respectively. In each of these examples, the signature of the genetic mutation is consistent with the misreplication of UV- induced DNA lesions. 1 Abbreviations: dPTP, pyrene triphosphate; TBE, Tris-HCl/borate/ EDTA; EDTA; ethylenediaminetetraacetate, sodium salt; dNTP, deoxy- The intuitive relationship between DNA damage and nucleoside triphosphate; dXTP, non-natural deoxynucleoside triphos- cancer has spawned significant interest in the elucidation of phate; 5-FITP, 5-fluoroindolyl-2′-deoxyriboside triphosphate; 5-AITP, 5-aminoindolyl-2′-deoxyriboside triphosphate; 5-NITP, 5-nitroindolyl- 2′-deoxyriboside triphosphate; 5-NapITP, 5-naphthylindolyl-2′-deox- † This research was supported by funding from the National Institutes yriboside triphosphate; 5-AnITP, 5-anthracene indolyl-2′-deoxyriboside of Health (CA118408) and the Skin Cancer Foundation to A.J.B. triphosphate; 5-PhITP, 5-phenylindolyl-2′-deoxyriboside triphosphate; * To whom correspondence should be addressed. Telephone: (216) 5-CE-ITP, 5-cyclohexene indolyl-2′-deoxyriboside triphosphate; 5-CH- 368-4723. Fax: (216) 368-3395. E-mail: [email protected]. ITP, 5-cyclohexylindolyl-2′-deoxyriboside triphosphate; gp43 exo-, ‡ Department of Chemistry. exonuclease-deficient mutant of the bacteriophage T4 DNA polymerase; § Department of Pharmacology. gp43 exo+, wild-type bacteriophage T4 DNA polymerase. 10.1021/bi602438t CCC: $37.00 © 2007 American Chemical Society Published on Web 03/23/2007 182 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4487 incorporation of various 5-modified indolyl nucleotides (Figure 1B) opposite a thymine dimer. These studies reveal that gp43 replicates thymine dimers and abasic sites via similar yet distinct mechanisms. This is best exemplified by the differences in the kinetic parameters measured for the incorporation of various non-natural nucleotides opposite either DNA lesion. In addition, this report evaluates the mechanisms for proofreading natural and non-natural nucle- otides paired opposite either DNA lesion. Distinct differences are observed using non-natural nucleotides such as 5-PhIMP which can be excised via exonuclease and pyrophosphoro- lytic activity when placed opposite an abasic site but not when placed opposite a thymine dimer. The collective data set is used to develop a comprehensive model highlighting the similarities and differences in the replication of a nontemplating lesion versus a bulky miscoding DNA adduct.

MATERIALS AND METHODS Materials.[γ-32P]ATP was purchased from M. P. Bio- Medicals (Irvine, CA). Ultrapure, unlabeled dNTPs were obtained from Pharmacia. Magnesium acetate and Trizma base were obtained from Sigma. Urea, acrylamide, and bisacrylamide were from Aldrich. The oligonucleotide containing a cis,syn thymine dimer was synthesized by TriLink Biotechnologies (San Diego, CA). All other oligo- nucleotides, including those containing a tetrahydrofuran ′ FIGURE 1: (A) Structures of 2 -deoxynucleoside triphosphates used moiety mimicking an abasic site, were synthesized by Operon or mentioned in this study. For convenience, dR is used to represent the deoxyribose triphosphate portion of the nucleotides. (B) Defined Technologies (Alameda, CA). Single-stranded and duplex DNA substrates used for kinetic analysis. X in the template strand DNA were purified and quantified as described previously denotes a thymine dimer, a thymine, or a tetrahydrofuran moiety (20). All other materials were obtained from commercial designed to functionally mimic an abasic site. sources and were of the highest available quality. Wild-type gp43 and the exonuclease-deficient mutant of gp43 (Asp- (15) in which the lesion lies outside the helical structure of 219 to Ala mutation) were purified and quantified as DNA to create a cavity that functionally resembles an abasic previously described (21, 22). The non-natural nucleotides used in this study were synthesized and purified as described site. - These observations lead us to question if similar mecha- previously (17 19). nisms are employed by other high-fidelity DNA polymerases Enzyme Assays. The assay buffer used in all kinetic studies during the replication of this and other bulky DNA lesions. consisted of 25 mM Tris-OAc (pH 7.5), 150 mM KOAc, As such, we performed a thorough kinetic analysis comparing and 10 mM 2-mercaptoethanol. All assays were performed the ability of the bacteriophage T4 DNA polymerase (gp43) at 25 °C. Polymerization reactions were monitored by to incorporate various natural and non-natural nucleotides analysis of the products on 20% sequencing gels (20). Gel opposite a thymine dimer versus an abasic site. We previ- images were obtained with a Packard PhosphorImager using ously demonstrated that non-natural analogues containing OptiQuant supplied by the manufacturer. Product formation significant π-electron surface areas such as 5-NITP, 5-PhITP, was quantified by measuring the ratio of 32P-labeled extended and 5-NapITP (Figure 1B) are incorporated ∼1000-fold more to nonextended primer. The ratios of product formation are efficiently than analogues such as 5-AITP and 5-CHITP corrected for substrate in the absence of polymerase (zero (Figure 1B) that have substantially smaller π-electron surface point). Corrected ratios are then multiplied by the concentra- areas (16-19). These results are consistent with a model in tion of the primer-template motif used in each assay to yield which the overall catalytic efficiency of nucleotide incor- total product. All concentrations are listed as final solution poration is directed by the base stacking capabilities of the concentrations. incoming nucleotide. In this model, the binding affinity of Determination of the Kinetic Rate and Dissociation the incoming nucleotide is linked with its shape and Constants for Incorporation of dXTP opposite a Thymine hydrophobicity, while the rate constant for incorporation of Dimer. In most cases, a rapid quench instrument (KinTek these non-natural nucleotides is solely dependent on the Corp., Clarence, PA) was used to monitor the time courses presence of π-electron density. for incorporation of a nucleotide opposite a thymine dimer. These results prompted us to test the following hypoth- Experiments were performed in which 250 nM 13/20TdT- esis: if gp43 processes a thymine dimer as a transient “abasic mer and variable concentrations of a nucleotide analogue site”, then analogues such as 5-NITP, 5-PhITP, and 5-NapITP (5-500 µM) were preincubated in assay buffer and mixed - are expected to be incorporated opposite either lesion with with 1 µM gp43 exo and 10 mM Mg(OAc)2. The reactions nearly identical kinetic parameters. This report outlines the were quenched with 500 mM EDTA at variable times structure-activity relationships derived from monitoring the (0.005-10 s) and analyzed as described above. Data obtained 183 4488 Biochemistry, Vol. 46, No. 15, 2007 Devadoss et al. for single-turnover DNA polymerization assays were fit to eq 1.

- y ) A(1 - e kt) + C (1) where A is the burst amplitude, k is the observed rate constant (kobs) for formation of an initial product, t is time, and C is a defined constant. Data for the dependency of kobs on dXTP concentration were fit to the Michaelis-Menten equation (eq 2) to provide values corresponding to kpol and KD. ) + kobs kpol[dXTP]/(KD [dXTP]) (2) FIGURE 2: Comparing the efficiency of insertion for non-natural d where kobs is the observed rate constant of the reaction, kpol and natural nucleoside triphosphates opposite a thymine dimer (T T) vs an abasic site (SP). Assays were performed as described in is the maximal polymerization rate constant, KD is the kinetic dissociation constant for dXTP, and dXTP is the concentra- the text. tion of the non-natural nucleotide substrate. V Acid ersus EDTA as the Quenching Agent. DNA (13/ Pyrophosphorolysis. DNA (14A/20T-mer, 14A/20TdT-mer, 20TdT, 250 nM) was incubated with KD concentrations of a or 14A/20SP-mer, 500 nM) was first incubated with 50 nM - non-natural nucleotide and 10 mM Mg(OAc)2. The reaction gp43 exo and 10 mM Mg(OAc)2. The reaction was then - was then initiated with 1 µM gp43 exo and quenched with initiated with 20 mM PPi and quenched with 500 mM EDTA either 500 mM EDTA or 1 M HCl at time intervals ranging at time intervals (5-300 s). The quenched samples were from 0.1 to 10 s using a rapid quench instrument as described processed as described above, and product formation was above. After the reaction had been quenched with 1 M HCl, analyzed using protocols similar to that used to monitor the 100 µL of a phenol/choloroform/isoamyl alcohol mixture was exonuclease activity of gp43 (23). added to extract the DNA polymerase, and the pH of the aqueous phase was neutralized by addition of ∼30 µLofa The pyrophosphorolytic activity of gp43 using 5-PhITP 1 M Tris/3 M NaOH mixture. Product formation was as the non-natural nucleotide was performed using a modified analyzed and quantified as described above. procedure. 13/20TdT-mer or 13/20SP-mer (500 nM) was Idle TurnoVer Measurements. DNA (13/20 d -mer or 13/ - T T incubated with 50 nM gp43 exo , 10 mM Mg(OAc)2, and 20SP-mer, 250 nM) was first preincubated with KD concentra- 10 µM 5-PhITP. In this case, the polymerase was allowed tions of 5-PhITP (40 µM) in the presence of 30 µM dCTP. to enzymatically incorporate 5-PhITP opposite the DNA Due to the nature of the DNA substrate (Figure 1A), insertion lesion to create the 14-mer. After ∼20 min (the time required of dCMP opposite G at position 13 in the template maintains to achieve >90% conversion of 13-mer to 14-mer), pyro- a usable primer-template motif for the insertion of the non- phosphorolysis was initiated with 20 mM PP . The reaction natural nucleotide opposite the thymine dimer lesion (position i was quenched through the addition of 500 mM EDTA at 14). In all cases, the reaction was initiated by the addition - of 1 µM gp43 exo+. Reactions were quenched with 500 mM time intervals (5 300 s) and the mixture processed as EDTA at time frames ranging from 5 to 600 s. The quenched described above. samples were processed, and product formation was analyzed as described previously (23). RESULTS AND DISCUSSION Exonuclease Degradation of Unmodified and Damaged DNA. Exonuclease reactions were performed under single- The series of natural and non-natural nucleotides illustrated turnover reaction conditions in which 250 nM DNA was in Figure 1B were used to probe for similarities or differences - preincubated with 10 mM Mg(OAc)2 in assay buffer, and in the ability of gp43 exo to replicate a thymine dimer + the reaction was initiated by adding 1 µM gp43 exo . These versus an abasic site. In either case, single-turnover condi- studies include monitoring of the enzymatic hydrolysis of tions were used in which 1 µM gp43 exo- was added last to the following DNA substrates: 14A/20TdT-mer, 14A/20SP- a preincubated solution of 50 µM dXTP and 500 nM DNA mer, 13/20TdT-mer, 13/20SP-mer, and 13/20T-mer. In all cases, (13/20 d -mer or 13/20 -mer). Results comparing incorpo- a rapid quench instrument was used to quench the reactions T T SP ration of a nucleotide opposite either DNA lesion are using 500 mM EDTA at time intervals ranging from 0.003 provided in Figure 2 and reveal significant differences in to 2 s. Degradation products were analyzed as described above, and data points were plotted as initial substrate (13- the efficiency of translesion DNA synthesis. For example, mer) remaining as a function of time. Data for each time although IndTP, 5-FITP, and 5-AITP are readily incorporated course were fit to eq 3 defining a first-order decay in initial opposite an abasic site, they are not efficiently incorporated substrate concentration. opposite the thymine dimer. The most striking difference, however, is that dATP and 5-NITP are poorly incorporated - y ) Ae kt + C (3) opposite the thymine dimer. This is noteworthy since dATP and 5-NITP are efficiently incorporated opposite an abasic where A is the amplitude of the burst phase, k is the observed site (Figure 2), and this result recapitulates previously rate constant for product formation, and C is the end point reported data (16, 24). It should also be noted that of the of the reaction. four natural nucleotides, only dATP exhibited any incorpora- 184 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4489 tion opposite the thymine dimer (data not shown).2 However, even at the highest tested concentration of dATP (1 mM), the measured rate constant of 0.007 s-1 is ∼21-fold slower -1 than the reported kpol value of 0.15 s measured for incorporation of dATP opposite an abasic site (24). In general, the difference in nucleotide utilization suggests that gp43 exo- replicates a thymine dimer via a mechanism different from that reported for an abasic site (16-19) and thus does not strictly obey the “A-rule” of translesion DNA synthesis. There are, however, instances in which gp43 exo- utilizes certain non-natural nucleotides with comparable efficiencies regardless of DNA lesion. As illustrated in Figure 2, indolyl analogues containing large π-electron surface areas such as 5-CEITP, 5-PhITP, and 5-NapITP are incorporated opposite the thymine dimer almost as effectively as an abasic site. As such, there is an apparent dichotomy in nucleotide utilization that cannot be rationalized solely by these qualita- tive data. The underlying reasons for these differences in nucleotide utilization were thoroughly investigated using a quantitative kinetic approach described below. Kinetic Parameters for Incorporation Opposite a Thymine Dimer. KD and kpol values were measured for the subset of non-natural nucleotides incorporated opposite the thymine dimer. All experiments were performed using single-turnover FIGURE 3: (A) Dependency of the apparent burst rate constant on the concentration 5-NapITP as measured under single-turnover conditions as previously described (16). Representative data conditions. Assays were performed using 1 µM gp43 exo-, 250 provided in Figure 3A show the time courses in incorporation nM 13/20 SP, 10 mM Mg(OAc)2, and 5-NapITP at variable of 5-NapITP opposite the thymine dimer. All time courses concentrations: 5 (b), 10 (0), 25 (3), 50 (+), and 100 µM(2). were fit to the equation for a single-exponential process to The solid lines represent the fit of the data to a single exponential. (B) The observed rate constants for incorporation (b) were plotted define kobs, the rate constant for product formation. As shown against 5-NapITP concentration and fit to the Michaelis-Menten in Figure 3B, the plot of kobs versus 5-NapITP concentration equation to determine values corresponding to KD and kpol. is hyperbolic, and a fit of the data to the Michaelis-Menten -1 equation yields a kpol value of 6.4 ( 0.5 s and a KD value in binding affinities of these analogues that are dependent of 13 ( 3 µM. Identical analyses were performed with other upon the nature of the lesion. For incorporation opposite an non-natural nucleotides, and the corresponding kpol, KD, and abasic site, the KD values of 5-CEITP, 5-PhITP, and kpol/KD values are summarized in Table 1. 5-NapITP are essentially identical at ∼15 µM(17-19). Inspection of Table 1 clearly indicates that analogues such During replication of a thymine dimer, however, these values as 5-CEITP, 5 PhITP, and 5-NapITP are preferentially vary significantly and appear to be linked with the overall incorporated opposite a thymine dimer. The catalytic ef- π-electron surface area of the incoming dXTP. Of these three ficiency (kpol/KD) for these analogues is between 2 and 4 analogues, 5-NapITP has the highest binding affinity (KD ) orders of magnitude greater than that for smaller analogues 13 µM) which coincides with its large π-electron surface such as 5-AITP and 5-FITP that lack extensive π-electron area (273 Å2). In contrast, the smaller π-electron surface area density. It is striking that the overall catalytic efficiency of 5-CEITP (180.7 Å2) arguably causes a weaker binding increases as the π-electron surface area increases. In general, affinity as manifest in the 10-fold higher KD value of 120 this variation reflects alterations in binding affinity rather µM. These differences are interesting since a correlation than perturbations in the polymerization rate constant since between binding affinity and π-electron surface area is not the kpol values for 5-CEITP, 5-PhITP, and 5-NapITP are observed during incorporation opposite an abasic site. ∼ - -1 nearly identical at 4 6s . The mechanistic significance Differences are also detected with respect to the kpol values of these observations is discussed below. which are again dependent upon the nature of the lesion. The favorable incorporation of analogues containing large kpol values measured with abasic site-containing DNA vary π-electron surface areas suggests that the thymine dimer can from 25 to 50 s-1, while they remain essentially invariant be processed as a noninstructional lesion since similar data during incorporation opposite a thymine dimer. The most were obtained for their incorporation opposite an abasic site dramatic effect, however, is that the rate constants for (17-19). However, the notable variations in kinetic param- incorporation are considerably slower opposite the thymine eters indicate that the misreplication of each lesion occurs dimer. The magnitude for this effect is largest with 5-PhITP via a distinct mechanism. One clear example is the difference in which the kpol for an abasic site is 12-fold faster than with the thymine dimer (53 and 4.4 s-1, respectively). 2 Incorporation of dATP opposite a thymine dimer occurs exclusively Perhaps the most unique kinetic behavior is again evident at the 3′-end of the lesion. Sequential insertion of dATP at both the 3′- when we evaluate the kinetic parameters for 5-NITP.3 The ′ and 5 -ends of the lesion does occur, but only at dATP concentrations K of 39 µM measured opposite the thymine dimer is only greater than 1 mM. However, the amount of product formed at the D 5′-end of the lesion is minimal and represents <2% of complete 2-fold higher than the value of 18 µM measured for an abasic turnover. site (16). Thus, the presence of π-electron density on the 185 4490 Biochemistry, Vol. 46, No. 15, 2007 Devadoss et al.

Table 1: Summary of Kinetic Rate and Equilibrium Constants Measured for the Insertion of dATP and 5-Substituted Indolyl-2′-deoxyriboside Triphosphates opposite a Thymine Dimer and an Abasic Sitea thymine dimer abasic site -1 -1 -1 -1 -1 -1 KD (µM) kpol (s ) kpol/KD (M s ) KD (µM) kpol (s ) kpol/KD (M s ) dATP NDb 0.007 ( 0.002c <20d 35 ( 5 0.15 ( 0.01 4600e 5-NITP 39 ( 19 0.042 ( 0.006 1100 18 ( 3 126 ( 6 7000000f 5-FITP NDb 0.030 ( 0.004c <60d 152 ( 41 0.30 ( 0.03 2000g 5-CHITP NDb 0.059 ( 0.004c <120d 6.2 ( 1.3 0.46 ( 0.003 74200h 5-CEITP 119 ( 31 4.4 ( 0.5 36980 4.6 ( 1.0 25.1 ( 1.5 5460000h 5-PhITP 36 ( 13 4.4 ( 0.43 122000 14 ( 353( 4 3800000g 5-NapITP 13 ( 3 6.4 ( 0.5 492300 10.3 ( 4.5 27.1 ( 1.5 2631100i 5-AnITP 31 ( 19 1.6 ( 0.5 51600 27 ( 7 5.3 ( 0.4 200000i a - The kinetic parameters kpol, KD, and kpol/KD were obtained under single-turnover reaction conditions using 500 nM gp43 exo , 250 nM 13/ 2+ b c 20TdT-mer, and 10 mM Mg at varying concentrations of non-natural nucleotide triphosphate (from 5 to 500 µM). Not determined. kobs values measured at the highest nucleotide concentration that was tested (500 µM or 1 mM). d Accurate values could not be determined since the lack of saturation kinetics prohibited the determination of true kpol and KD values. Thus, the reported catalytic efficiencies reflect upper estimates based e upon the rate constant (kobs) measured using 500 µM dXTP divided by the highest nucleotide concentration that was tested (500 µM). Values taken from ref 24. f Values taken from ref 16. g Values taken from ref 17. h Values taken from ref 19. i Values taken from ref 18.

Table 2: Summary of Kinetic Rate and Equilibrium Constants Measured for the Incorporation of dATP and 5-Substituted Indolyl-2′-deoxyriboside Triphosphates opposite a Thymine Dimer or Thyminea thymine dimer thymine -1 -1 -1 -1 -1 -1 KD (µM) kpol (s ) kpol/KD (M s ) KD (µM) kpol (s ) kpol/KD (M s ) dATP NDb 0.007 ( 0.002 c <20d 10 ( 0.5 100 ( 10 10000000e 5-NITP 39 ( 19 0.042 ( 0.006 1100 9 ( 1 0.9 ( 0.1 100000f 5-FITP NDb 0.030 ( 0.004 c <60d 141 ( 32 0.040 ( 0.003 2900g 5-CHITP NDb 0.059 ( 0.004c <120d 25 ( 10 0.018 ( 0.005 720h 5-CEITP 119 ( 31 4.4 ( 0.5 36980 63 ( 12 0.076 ( 0.005 1120h 5-PhITP 36 ( 13 4.4 ( 0.4 122000 25 ( 7 0.16 ( 0.01 6400g 5-NapITP 13 ( 3 6.4 ( 0.5 492300 16 ( 8 2.21 ( 0.4 135600i 5-AnITP 31 ( 19 1.6 ( 0.5 51600 29 ( 15 0.53 ( 0.11 18600i

a - The kinetic parameters kpol, KD, and kpol/KD were obtained under single-turnover reaction conditions using 500 nM gp43 exo , 250 nM 13/ 2+ b c 20TdT-mer, and 10 mM Mg at varying concentrations of non-natural nucleotide triphosphate (from 10 to 500 µM). Not determined. kobs values measured at the highest nucleotide concentration that was tested (500 µM or 1 mM). d Accurate values could not be determined since the lack of saturation kinetics prohibited the determination of true kpol and KD values. Thus, the reported catalytic efficiencies reflect upper estimates based e upon the rate constant (kobs) measured using 500 µM dXTP divided by the highest nucleotide concentration that was tested (500 µM). Values taken from ref 20. f Values taken from ref 16. g Values taken from ref 17. h Values taken from ref 19. i Values taken from ref 18. incoming nucleotide appears to influence binding affinity. Of all of the non-natural nucleotides tested in this study, -1 In contrast, the kpol value of 0.042 s measured using the only 5-AnITP displays nearly identical kinetic parameters -1 thymine dimer is 3000-fold lower than the value of 126 s regardless of DNA lesion. For example, the KD value of 31 measured for an abasic site (16). This difference suggests µM opposite a thymine dimer is identical to that of 27 µM that the presence of π-electron density alone is insufficient measured opposite an abasic site (18). Furthermore, the kpol to facilitate the conformational change that limits nucleotide value of 1.6 s-1 for incorporation opposite a thymine dimer -1 incorporation. In fact, it appears that the kpol values for is only 3-fold slower than the value of 5.3 s reported using incorporation opposite the thymine dimer depend equally an abasic site (18). While these data are consistent with the upon contributions from both the π-electron density and the aforementioned hypothesis, it should be noted that 5-AnITP overall size of the analogue. The latter half of this finding is also displays unique kinetic parameters for insertion opposite reminscent of the shape-complementarity model originally templating nucleobases. Specifically, the KD of 29 µM for proposed by Kool and co-workers (reviewed in ref 25). incorporation opposite thymine (18) is identical to that of 31 µM for incorporation opposite a thymine dimer. Likewise, -1 3 Initial data reported in Figure 2 indicated that 5-NITP is poorly the kpol of 0.5 s is only 3-fold slower than the value of 1.6 -1 incorporated opposite a thymine dimer. However, this low efficiency s measured for incorporation opposite a thymine dimer partially reflects the reaction conditions employed in this experiment, (18). i.e., low dXTP concentration and a short reaction time of 10 s. In fact, increasing the concentration of 5-NITP and monitoring the reaction at These kinetic data raised the possibility that a thymine time frames encompassing 15-600 s resulted in a dose- and time- dimer may simply be replicated as a templating nucleobase dependent increase in the level of product formation (Supporting rather than as an abasic site. This was evaluated by Information). From these experiments, a KD of 39 ( 19 µM and a kpol value of 0.042 ( 0.006 s-1 were measured. Similar experiments were comparing the kinetic parameters for these non-natural performed using other analogues such as 5-FITP and 5-CH-ITP. In nucleotides opposite a thymine dimer versus an unmodified both cases, the rate and amount of primer elongation increased with thymine (Table 2). Indeed, certain analogues lacking large the concentration of nucleotide. However, saturation kinetics were not π-electron surface areas (5-NITP, 5-FITP, and 5-CH-ITP) observed even at the maximal concentration (500 µM) tested for either non-natural nucleotide. Hence, the lack of saturation prevented accurate are incorporated far more efficiently opposite T than the measurements of kpol and KD values. cross-linked DNA adduct. However, other analogues such 186 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4491

FIGURE 4: (A) Proposed model for the enzymatic incorporation of non-natural nucleotides. The first represents the binding of dNTP to the polymerase-DNA complex (KD). After nucleotide binding, the polymerase undergoes a conformational change (kpol) that is required to place the triphosphate moiety in the proximity of the positively charged amino acids as well as to stack the nucleobase portion of the incoming dNTP into the hydrophobic environment of the interior of the duplex DNA. The final stage of the catalytic cycle is phosphoryl transfer step that is required for elongation of the primer strand (kchem). Panels B-E provide computer-generated models for DNA containing an abasic site (B and C) or a thymine dimer (D and E). All models were constructed using Spartan ’04. (B) Computer-generated model for the structure of DNA containing an abasic site. (C) 5-Phenylindoledeoxyribose monophosphate paired opposite an abasic site in which the non-natural nucleobase is placed in an intrahelical conformation. (D) Computer-generated model for the structure of DNA containing a thymine dimer. (E) 5-Phenylindoledeoxyribose monophosphate paired opposite a thymine dimer in which the non-natural nucleobase is placed in an intrahelical conformation. as 5-CEITP, 5-PhITP, and 5-NapITP that contain large site or a templating thymine. Instead, the data argue that the π-electron surface areas exhibit an opposite trend as they bulky lesion is replicated as a “hybrid” containing features are incorporated more effectively opposite a thymine dimer common to both the nontemplating lesion and thymine. To compared to thymine. In these latter instances, the increased explain such a phenomenon, we use the model illustrated in efficiency is caused by faster kpol values rather than by an Figure 4A to propose a unified mechanism for replicating enhancement in binding affinity. unmodified and damaged DNA. In the case of normal DNA These differences are significant as they suggest that gp43 replication, we propose that the templating nucleobase is does not replicate the thymine dimer strictly as an abasic oriented in an extrahelical position that creates a transient 187 4492 Biochemistry, Vol. 46, No. 15, 2007 Devadoss et al.

“void” mimicking an abasic site (19). This hypothesis is based upon kinetic evidence using our non-natural nucle- otides (16-19) and structural models of various DNA polymerases bound to nucleic acid (26-28). Our kinetic data indicate that large, bulky non-natural nucleotides can easily fill the void produced by this transient intermediate. In fact, the low KD values measured for their insertion opposite a true abasic site provide further evidence of this mechanism. As seen in panels B and C of Figure 4, the non-natural nucleobase of 5-PhITP can fill the void at an abasic site and is stabilized by π-π electron interactions with the penulti- mate base pair. FIGURE 5: Time courses for the incorporation of 5-PhITP opposite Unlike binding affinity, however, kpol values are highly a thymine dimer using EDTA (9) or HCl (O) as the quenching - dependent upon the presence of a templating nucleobase. reagent. gp43 exo (1 µM) and 5′-labeled 13/20TdT-mer (250 nM) 2+ When a normal templating base is present, the kpol values were preincubated, mixed with 10 mM Mg and 30 µM 5-PhITP are slow since the shear bulk of large non-natural nucleotides to initiate the reaction, and quenched with either 500 mM EDTA or 1 M HCl at variable times (0.05-5 s). After the reaction had hinders the facile repositioning of the templating nucleobase been quenched with HCl, 100 µL of a phenol/chloroform/isoamyl from an extrahelical into an intrahelical conformation. In alcohol mixture was added to extract the polymerase, and the pH contrast, the kpol values at an abasic site are significantly of the aqueous phase was neutralized with the addition ofa1M faster since the lack of a templating nucleobase circumvents Tris/3 M NaOH mixture. Product formation was analyzed by denaturing gel electrophoresis followed by phosphorimaging analy- the need for repositioning. Furthermore, the rate of the -1 sis. An amplitude of 211 ( 6 nM and a kobs of 2.51 ( 0.25 s conformational change step is dependent upon the presence were obtained using EDTA as the quench, while an amplitude of -1 of π-electron density (19) and is consistent with the favorable 220 ( 9 nM and a kobs of 0.99 ( 0.15 s were obtained using stacking interactions of the non-natural nucleobase within HCl as the quenching agent. the void of the abasic site (Figure 4C). The dynamics of replication of a thymine dimer are to achieve base pairing interactions. These possibilities await consistent with this model, especially if one considers that the results of our ongoing structural studies of various non- this lesion contains physical features common to both a natural nucleotides paired opposite damaged and nondam- templating base and a nontemplating abasic site. In this aged DNA. model, the 3′-T of the lesion exists predominantly, but not Rate-Limiting Step for Replication of a Thymine Dimer. entirely, in an extrahelical position while the 5′-T of the To further compare and contrast the mechanism of translesion lesion remains in an intrahelical position. As shown in Figure DNA synthesis, a series of experiments were performed to 4D, the extrahelical positioning of the 3′-T creates an evaluate the rate-limiting step during replication opposite a intermediate resembling an abasic site. However, the covalent thymine dimer. Previous studies using denaturing versus bond between the intrahelical 5′-T and the extrahelical 3′-T nondenaturing quenching systems demonstrated that the hinders the overall mobility of the lesion such that the rate conformational change preceding phosphoryl transfer is rate- constant for the prerequisite conformational change step limiting for the incorporation of non-natural nucleotides preceding phosphoryl transfer is significantly slower com- opposite an abasic site (16, 19, 24). A similar approach was pared to that for a true abasic site. used here to monitor time courses in nucleotide incorporation These models also indicate that the unique shape of the using EDTA (nondenaturing quench) versus HCl (denaturing thymine dimer influences ground-state binding. For example, agent). Any observed differences in the amount and/or rate the large bulky analogue, 5-CHITP, is most likely sterically constants in product formation using these agents can provide hindered from binding in a proper orientation, and this is information regarding the existence of various enzyme forms, ‚ ‚ ‚ ′‚ ‚ reflected in its poor binding affinity (KD > 500 µM). In including E DNA, E DNA dXTP, and E DNA dXTP, that contrast, analogues containing large, flat aromatic systems accumulate before the phosphoryl transfer step (E′‚DNAn+1‚ (5-PhITP and 5-NapITP) can interact more stably within the PPi). smaller void caused by the 3′-T (Figure 4E). Therefore, Initial experiments focused on the incorporation of 5-PhI- binding affinity increases as the stacking interactions between TP opposite the thymine dimer.4 Time courses generated the incoming nucleotide and DNA become optimal. using KD concentrations of 5-PhITP (40 µM) are provided We note that the aforementioned model has not been in Figure 5 and reveal that the amplitude in product formation proven and that other models may exist to explain the is independent of quenching agent (210 nM using either HCl observed differences in nucleotide utilization. A potential or EDTA). The identity in burst amplitudes indicates that argument against our proposed model is the assumption that phosphoryl transfer is not rate-limiting for incorporation of the substituent group at the C5 position of the indole ring is 5-PhITP opposite the lesion. However, the difference in rate directed toward the opposing strand such that stacking constants with EDTA and HCl (2.5 and 1 s-1, respectively) interactions occur with the penultimate bases on both the indicates that incorporation opposite the thymine dimer is primer and template strands. An alternative model is one in limited by the conformational change preceding phosphoryl which these non-natural nucleotides exist in the syn confor- transfer. A similar conclusion was made after the incorpora- mation such that the C5 position of the indole ring is directed tion of 5-PhITP opposite an abasic site was monitored (17). away from the opposing strand. Another possibility is that the indole residue simply intercalates between the penulti- 4 Similar experiments could not be performed using dATP since this mate and templating base as opposed to acting as a surrogate natural nucleotide is poorly incorporated opposite the thymine dimer. 188 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4493 HCl is reduced 40% compared to that with EDTA (Figure 6C). Furthermore, the rate constant using HCl is 3.5-fold slower than measured with EDTA (0.018 and 0.063 s-1, respectively). The reduced rate constant and 40% lower amplitude indicate that the conformational change and the phosphoryl transfer step contribute equally to limiting the overall rate constant for incorporation. These results are again interpreted with respect to the models provided in Figure 4. We propose that the confor- mational change step represents structural reorganization of the incoming nucleotide with the primer-template. During translesion DNA synthesis, it is easy to envision that the non-natural nucleotide can fill the void at either an abasic site or a thymine dimer and is properly oriented for phosphoryl transfer. This is not the case during incorporation opposite a templating nucleobase since both the conforma- tional change and phosphoryl transfer steps are rate-limiting for the insertion of bulky analogues such as 5-CHITP. It is most likely that this bulky nucleobase hampers the reposi- tioning of the primer-template which also influences the rate of the phosphoryl transfer step. Exonuclease Proofreading ActiVity at a Thymine Dimer. The bacteriophage T4 polymerase possesses a vigorous exonuclease activity (20) that plays a significant role in maintaining fidelity (29). Previous work demonstrated that gp43 removes dAMP paired opposite an abasic site ∼30- fold faster than when dAMP is properly paired with thymine (23). The faster rate constant was proposed to reflect an increase in exonuclease activity caused by distortion of the primer-template through inappropriate hydrogen bond- ing and stacking interactions at an abasic site. This mech- FIGURE 6: Time courses for the incorporation of 5-CH-ITP opposite a thymine dimer (A), an abasic site (B), or thymine (C) using EDTA anism led to the following hypothesis: if both DNA lesions (b) or HCl (O) as the quenching agent. Assays were performed as are functionally identical, i.e., both are noninstructional, then described in the legend of Figure 5 and the text. The following the kinetics of mispair excision at a thymine dimer should values were obtained for incorporation opposite a thymine dimer: be indistinguishable from that of an abasic site. This ) ( ) ( -1 amplitude 360 10 nM and kobs 0.060 0.009 s using hypothesis was evaluated by comparing the kinetics by which EDTA, and amplitude ) 360 ( 15 nM and kobs ) 0.031 ( 0.003 + s-1 using HCl. The following values were obtained for incorporation gp43 exo excises natural and non-natural nucleotides paired opposite an abasic site: amplitude ) 400 ( 10 nM and kobs ) 2.5 opposite a thymine dimer, an abasic site, or a templating -1 ( 0.3 s using EDTA, and amplitude ) 360 ( 20 nM and kobs ) thymine. 2.6 ( 0.3 s-1 using HCl. The following values were obtained for (a) RemoVal of Natural Nucleotides. Reactions monitoring incorporation opposite thymine: amplitude ) 420 ( 10 nM and ′ -1 the enzymatic hydrolysis of dAMP paired opposite the 3 - kobs ) 0.063 ( 0.007 s using EDTA, and amplitude ) 250 ( 50 -1 end of a thymine dimer or an abasic site were performed by nM and kobs ) 0.018 ( 0.008 s using HCl. employing single-turnover conditions in a rapid quench Therefore, the conformational change preceding phosphoryl instrument as described previously (23). The time courses transfer still remains the rate-limiting step regardless of for excision of dAMP from either lesion are superimposable differences in the kinetic parameters measured for incorpora- and best defined as a single-exponential decay (Supporting tion of 5-PhITP opposite either lesion. Information). Although the measured rate constants are To further evaluate this mechanism, we next monitored identical, they are ∼10-fold faster than the rate constant for the incorporation of 5-CHITP opposite a thymine dimer, an excising dAMP paired opposite thymine (data not shown). abasic site, and thymine using the different quenching agents. The faster rate constants are likely caused by distortion of As shown in Figure 6A, the amplitude for incorporation of the primer-template junction induced by either lesion and 5-CHITP opposite the thymine dimer is independent of suggest that the thymine dimer does indeed resemble an quenching agent. However, the rate constant measured using abasic site. We propose that mispairs formed at either DNA EDTA is 2-fold faster than that using HCl (0.06 and 0.03 lesion enhance the enzyme’s ability to partition the mis- s-1, respectively). The difference in rate constants again aligned primer from the polymerase active site into its indicates that the conformational change preceding phos- exonuclease domain. phoryl transfer is the rate-limiting step. To provide further evidence of DNA partitioning, we Identical experiments performed by monitoring incorpora- attempted to measure the idle turnover activity of the tion of 5-CHITP opposite the abasic site (Figure 6B) show polymerase. Idle turnover is a process in which the poly- identical burst amplitudes and rate constants in product merase incorporates a dNTP and then excises the inserted formation. In marked contrast, the amplitude of product dNMP in the absence of the next required nucleotide formation for incorporation of 5-CHITP opposite T using triphosphate (30). Unfortunately, attempts to monitor the idle 189 4494 Biochemistry, Vol. 46, No. 15, 2007 Devadoss et al.

FIGURE 8: Comparison of idle turnover kinetics for insertion of + FIGURE 7: Primer degradation of DNA containing a thymine dimer 5-PhITP opposite an abasic site vs a thymine dimer. gp43 exo (1 (b), an abasic site (×), or thymine (0). All experiments were µM) was added last to a solution containing 250 nM 5′-labeled + 2+ performed by mixing a preincubated solution of 1 µM gp43 exo 13/20SP-mer (O) or 13/20TdT-mer (b), 10 mM Mg , and 40 µM and 10 mM Mg2+ with 250 nM 5′-labeled DNA and 10 mM Mg2+ 5-PhITP. Reactions were terminated by the addition of 500 mM (final concentrations) and terminating the reaction at various times EDTA at time intervals ranging from 50 to 600 s. Nucleotide by the addition of 350 mM EDTA. Each time course represents an incorporation and excision were analyzed by denaturing gel average of two independent determinations. Time courses were fit electrophoresis. to the equation for single-exponential decay, y ) Ae-kt + C, where A is the burst amplitude, k is the observed rate constant for product polymerase cannot incorporate opposite the lesion which formation, and C is the end point of the reaction. The rate constant causes degradation of the 14-mer via exonuclease activity. ( -1 for the degradation of 13/20TdT-mer is 2.0 0.1 s . The rate Idle turnover of 5-PhITP at a thymine dimer is markedly constants for the degradation of 13/20T-mer and 13/20SP-mer are 0.74 ( 0.09 and 0.82 ( 0.07 s-1, respectively. different as the steady-state phase for 14-mer accumulation is significantly longer than that measured opposite an abasic site. The accumulation of 14-mer reflects the inability of gp43 turnover of dATP at the thymine dimer were futile since to excise the non-natural nucleotide when it is paired opposite gp43 poorly incorporates this natural nucleotide opposite the the thymine dimer. The difference in degradation is arguably lesion (vide supra). As an alternative, we directly measured caused by the ability of the non-natural nucleotide to stack the rate constant for excision of dAMP from the penultimate into a dead-end complex at a thymine dimer but not at an base pair relative to the DNA lesion using 13/20T-mer, 13/ abasic site. The models illustrated in panels C and E of Figure 20SP-mer, and 13/20TdT-mer as substrates. Time courses 4 are again useful in providing insight into the dichotomy provided in Figure 7 reveal that the exonuclease activity of in exonuclease activity. At an abasic site (Figure 4C), the gp43 is 3-fold faster with DNA containing a thymine dimer 5-phenylindole moiety stacks very well in an intrahelical compared to DNA containing an abasic site or an unmodified conformation and conforms satisfactorily to the overall shape thymine. The increased exonuclease activity suggests that and size of a natural Watson-Crick base pair (17). Thus, gp43 “senses” the bulky DNA lesion and facilitates partition- the enzyme excises the non-natural nucleotide at a rate ing of the DNA from the polymerase site into the exonuclease constant comparable to that of excision of dAMP opposite active site at a higher frequency.5 T(23). Figure 4E also shows that 5-phenylindole can stack (b) RemoVal of Non-Natural Nucleotides. Idle turnover within the void created by the thymine dimer. However, the experiments measured the proofreading capability of gp43 shape of the 5-phenylindole‚thymine dimer mispair does not with non-natural nucleotides paired opposite the thymine accurately mimic the overall shape and size of a natural base dimer.6 This activity was quantified as previously described pair. At face value, one would predict that this distortion (23) using a modified gel electrophoresis protocol monitoring would cause the polymerase to degrade the mispair very the amount of extension (13-mer to 14-mer) and subsequent efficiently. Indeed, we demonstrated earlier that gp43 easily excision (14-mer to 13-mer) of the DNA as a function of excises dAMP when paired opposite a thymine dimer due time. Figure 8 provides representative time courses compar- to the distortion of the primer-template. However, the ing idle turnover of 5-PhITP opposite either DNA lesion. inability to excise 5-PhIMP argues against a model invoking At an abasic site, the primer is rapidly elongated to form simple distortion of the primer-template. We hypothesize 14-mer. This burst is followed by a short steady-state phase that the proximity of the 5′-T of the lesion to the phenyl of product accumulation that defines the kinetics of idle moiety of the non-natural nucleotide induces aberrant stack- turnover. During the process of nucleotide incorporation and ing interactions that can inhibit the vigorous proofreading excision, the concentration of 5-PhITP decreases until it capabilities of gp43. Further investigations are currently becomes lower than the KD value. At this point, the underway to validate this potential model. RemoVal of Nucleotides Via Pyrophosphorolysis. Pyro- 5 Additional support for this mechanism comes from experiments phosphorolysis, the reversal of DNA polymerization, is an monitoring product formation under different preincubation conditions important enzyme activity that can remove nucleotides from (Supporting Information). Briefly, the rate constant in product formation nonextendable primers in the absence of exonuclease proof- is 3-fold slower when gp43 is preincubated with 13/20TdT-mer than reading activity (31). Reverse transcriptases and other viral when gp43 is preincubated with nucleotide or added last to initiate the reaction. This phenomenon is unique to the presence of the thymine polymerases use pyrophosphorolysis to remove various dimer as identical time courses in product formation are obtained using chain-terminating nucleotide analogues from their genomic unmodified or abasic site-containing DNA regardless of preincubation material (reviewed in ref 32). This activity is typically conditions (data not shown and ref 24). 6 associated with the development of drug resistance to Idle turnover measurements can be performed with non-natural - nucleotides such as 5-PhITP as its catalytic efficiency for incorporation nucleoside analogues such as AZT and ddI (33 35). In this is high (>105 M-1 s-1) for an abasic site or thymine dimer. report, we quantified the pyrophosphorolytic activity of gp43 190 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4495 provided in panels C and E of Figure 4. At an abasic site, the 5-phenylindole moiety stacks well opposite the lesion and mimics the overall shape and size of a natural Watson- Crick base pair (17). Thus, gp43 binds this mispair in the polymerase active site and is poised for elongation. However, in the absence of the next correct dNTP, the polymerase catalyzes pyrophosphorolysis when supplied with PPi. Note that the rate of removal of 5-PhIMP opposite an abasic site is ∼8-fold slower than that measured for the pyrophospho- rolysis of dATP paired opposite T (Figure 9B). The differ- ence in rates could reflect the enhanced stability of the mispair through base stacking interactions of 5-PhIMP or the presence of an altered conformation that is not optimal for pyrophosphorolysis. Regardless, it is clear that the non- natural nucleotide is processed more effectively when paired opposite an abasic site compared to a thymine dimer. Conclusions. UV radiation causes a variety of covalently modified DNA lesions, the most prevalent form of which is the cis,syn thymine dimer (reviewed in ref 3). The hydrogen bonding groups required for base pair recognition are not - FIGURE 9: Pyrophosphorolysis activity of gp43 exo on unmodified altered at a thymine dimer, and this predicts that the adduct or damaged DNA templates. (A) Time courses for the excision of should be replicated as a miscoding lesion. However, thymine dAMP paired opposite T (b), dAMP paired opposite an abasic site dimers also induce deformations in the helical structure of 0 × ( ), dAMP paired opposite a thymine dimer ( ), 5-PhIMP paired DNA (36) that create a cavity resembling an abasic site, a opposite an abasic site (O), and 5-PhIMP paired opposite a thymine dimer (1). Each time course represents an average of two noninstructional DNA lesion. In this report, we used our independent determinations. (B) Comparison of the rates of series of non-natural nucleotides to demonstrate that the high- pyrophosphorolysis for excision of a nucleotide opposite unmodified fidelity bacteriophage T4 DNA polymerase does indeed or damaged DNA templates. Rates were determined from the linear replicate the bulky lesion like an abasic site. Analogues phase of the time courses provided in panel A. containing large π-electron surface areas are incorporated opposite either lesion ∼1000-fold more efficiently than those to evaluate its potential role in excising natural and non- analogues devoid of π-electron density. One striking example natural nucleotides paired opposite normal or damaged is that the catalytic efficiency for incorporation of 5-PhITP templating nucleobases. Assays monitoring the excision of opposite a thymine dimer is ∼6000-fold higher than that for dAMP were performed under pseudo-first-order reaction the incorporation of dATP. Similar results were obtained with conditions using 500 nM DNA, 50 nM gp43 exo-, and 20 the bacteriophage T7 DNA polymerase using dPTP as the mM pyrophosphate (PP ). As shown in Figure 9A, gp43 exo- i non-natural nucleotide (13). However, the selectivity for efficiently removes dAMP paired opposite T through pyro- incorporation of dPTP versus dATP opposite the thymine phosphorolysis and recapitulates data previously reported by dimer is minimal since the reported value of ∼15 (13)is Capson et al. (20). This datum infers that the normal primer- significantly lower that that of ∼6000 measured here using template motif is in a proper conformation that allows gp43 5-PhITP and the T4 DNA polymerase. Regardless, it appears to bind it in the polymerization active site. Although the that both high-fidelity DNA polymerases use similar mech- enzyme is poised for elongation, the absence of the next anisms to replicate a thymine dimer. correct dNTP allows the enzyme to use PP as a substrate to i There are, however, notable differences in the kinetic reverse the polymerization reaction. parameters for these non-natural nucleotides that provide Pyrophosphorolysis activity is not observed when dATP evidence that gp43 does not replicate a thymine dimer is paired opposite either an abasic site or a thymine dimer identically as an abasic site. Figure 10 provides comparative (Figure 9A). This result is intriguing since both mispairs structure-activity relationships for the incorporation of activate the exonuclease activity of gp43 (vide supra). The several non-natural nucleotides opposite a thymine dimer dichotomy in activity likely reflects the inability of gp43 to versus an abasic site. During incorporation opposite an abasic properly bind these mispairs in the polymerization active site. site, it is clear that each of these nucleotide analogues can We argue that the enzyme partitions these mispairs into the stack within the void present at the lesion. We argue that exonuclease active site of gp43 and would be consistent with these stacking interactions account for the relatively equal the enhanced exonuclease activity of gp43 when dAMP is KD values of these analogues at an abasic site (16-19). For paired opposite the 3′-T of the thymine dimer (vide supra). insertion opposite an abasic site, binding affinity is inde- Partitioning of the mispair into the exonuclease active site pendent of π-electron density. Therefore, the lower catalytic would also explain the reluctance of the polymerase to extend efficiency for 5-CHITP compared to those for 5-CEITP, beyond the mispair by incorporating opposite the 5′-T of 5-PhITP, and 5-NITP results from a low kpol value caused the thymine dimer. by the diminished π-electron surface area of 5-CHITP. As With this in mind, it is fascinating that 5-phenylindole can before, we argue that π-electron surface area plays the be removed by pyrophosphorolysis when placed opposite an preeminent role in enhancing the rate of the conformational abasic site but not when it is paired opposite a thymine dimer. change step preceding phosphoryl transfer (16-19). As These results have again been interpreted with the models implied within the model provided in Figure 10, the 191 4496 Biochemistry, Vol. 46, No. 15, 2007 Devadoss et al.

FIGURE 10: Proposed structure-activity relationships for the enzymatic incorporation of various non-natural nucleotides opposite a thymine dimer vs an abasic site. All models were constructed using Spartan ’04 and are designed to illustrate the influence of π-electron surface area, shape, and size on the overall catalytic efficiency for incorporation opposite either DNA lesion. Please refer to text for further details and discussion.

π-electron surface areas of 5-CEITP, 5-PhITP, and 5-NITP likely reflects the enzyme’s ability to partition the misaligned are also important for stacking interactions with the penul- primer from the polymerase active site into its exonuclease timate base pair. domain. Similarities are also observed in the pyrophospho- These theoretical models can also be used to explain the rolysis activity of gp43. The enzyme does not process dATP differences in kinetic behavior at the thymine dimer. The when the natural nucleotide is paired opposite either an abasic catalytic efficiency for incorporation opposite the thymine site or a thymine dimer. The lack of pyrophosphorolysis at dimer is similar to that for an abasic site as both are linked these naturally occurring mispairs suggests that the primer- with the overall π-electron surface area of the incoming template is partitioned away from the polymerization domain. nucleotide. However, the rate constants for the conforma- The ability to partition DNA away from the polymerization tional change step at a thymine dimer are considerably slower domain and into the exonuclease active site could contribute compared to those for an abasic site. We interpret these to the lack of extension beyond either class of mispair. differences to reflect the influence of steric hindrance In this respect, both thymine dimers and abasic sites are imposed by the bulky lesion. In the case of 5-CHITP, it is considered to be strong replication blocks that lead to arrests clear that the shear bulk of the incoming nucleotide prevents in DNA synthesis (37-39). Their ability to inhibit high- optimal interactions with the bulky thymine dimer. In addition, the effect of steric hindrance diminishes as the shape fidelity DNA polymerases is proposed to reflect steric of the 5-substituent group becomes more planar as the degree constraints imposed by the “tightness” of the active site of of π-electron density increases (compare 5-CEITP vs 5-PhI- this family of high-fidelity DNA polymerases (40). The data TP opposite the thymine dimer). One exception is 5-NITP provided here are consistent with this mechanism as geo- as it is poorly incorporated opposite the thymine dimer. metrical constraints imposed by covalent linkage of two Although 5-NITP is relatively planar, the model provided adjacent thymines prohibit the lesion from properly fitting in Figure 10 indicates that it preferentially interacts with the within the polymerase’s active site. From a biological 5′-end of the thymine dimer as opposed to stacking within perspective, the inability of high-fidelity polymerases to the void caused by the thymine dimer. This difference may efficiently incorporate dNTPs opposite either DNA lesion account for the poor incorporation of 5-NITP opposite the is undoubtedly an important step in preventing translesion bulky lesion. Collectively, these analyses indicate that synthesis. However, we propose that the capacity of the DNA biophysical parameters influencing binding affinity and the polymerase to partition mispairs away from its polymeriza- rate of the conformational change step are differentially tion domain has equally important ramifications on the influenced by the dynamic features of the DNA lesion. biological outcome of translesion DNA replication. In Despite differences in polymerization activity, we provide prokaryotes and eukaryotes, DNA replication blocks induced evidence that each lesion is processed similarly with respect by diverse DNA lesions can be rescued through the action to exonuclease proofreading and pyrophosphorolysis. The of various error-prone DNA polymerases (41-46). Relevant kinetics for excision of dAMP from either DNA lesion are examples include pol η and Dpo4 which are error-prone identical and significantly faster than the excision of dAMP polymerases possessing “loose” active sites (14, 47) that can paired opposite an unmodified thymine. This enhancement bypass thymine dimers (47, 48). Paradoxically, the activity 192 Incorporation of Non-Natural Nucleotides Biochemistry, Vol. 46, No. 15, 2007 4497 of error-prone polymerases at damaged DNA ultimately induction of an abasic site, T-T (6-4) photoadduct and T-T cis- allows the cell to maintain genomic fidelity. However, to syn cyclobutane dimmer, Genetics 169, 575-582. 8. Besaratinia, A., Synold, T. W., Xi, B., and Pfeifer, G. P. 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