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Probing the Base Stacking Contributions During 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 _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *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
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