University of Connecticut OpenCommons@UConn
Doctoral Dissertations University of Connecticut Graduate School
1-7-2020
Chemical Biology of 6-Nitrochrysene Induced Deoxyadenosine DNA Adduct and Formamidopyrimidine DNA Lesion: Mutagenesis and Genotoxicity in E. coli and Human Cells
Brent V. Powell University of Connecticut, [email protected]
Follow this and additional works at: https://opencommons.uconn.edu/dissertations
Recommended Citation Powell, Brent V., "Chemical Biology of 6-Nitrochrysene Induced Deoxyadenosine DNA Adduct and Formamidopyrimidine DNA Lesion: Mutagenesis and Genotoxicity in E. coli and Human Cells" (2020). Doctoral Dissertations. 2398. https://opencommons.uconn.edu/dissertations/2398 Chemical Biology of 6-Nitrochrysene Induced Deoxyadenosine DNA Adduct and Formamidopyrimidine DNA Lesion: Mutagenesis and Genotoxicity in E. coli and Human Cells
Brent Valentine Powell, Ph.D.
University of Connecticut, 2020
The environmental pollutant, 6-nitrochrysene (6-NC) is the most potent carcinogen evaluated by the newborn mouse assay. The genotoxicity of 6-NC is derived from its ability to form electrophilic species in cells which can react with nucleophilic sites of 2'-deoxyguanosine (dG) and 2’-deoxyadenosine (dA) in DNA to generate DNA-carcinogen adducts. DNA lesions derived from 6-NC can play important roles in the development of human cancer by inducing mutations in crucial genes resulting in the disruption of gene expression. Mutations in an oncogene, a tumor- suppressor gene such as p53, or a gene that controls the cell cycle can lead to uncontrolled cell growth, resulting in carcinogenesis, a process which ultimately gives rise to human cancer. Most of these mutations arise through error-prone mutagenic bypass of the lesions which is enabled by low fidelity translesion synthesis (TLS) DNA polymerases.
6-NC is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields major DNA adducts at the C8 and N2 positions of dG, N-(dG-8-yl)-6-AC and 5-(dG-N2-yl)-6-AC. The nitroreduction pathway also yield adduct at the C8 position of dA, N-(dA-8-yl)-6-AC and a 2'-deoxyinosine (dI) adduct, N-(dI-8- yl)-6-AC; which is believed to be a product of deamination of the adenine adduct N-(dA-8-yl)-6-
AC. The N-(dA-8-yl)-6-AC adduct is particularly interesting, a nucleotide excision repair assay demonstrated that it is repaired much more slowly than many other bulky DNA adducts, including other DNA adducts formed by 6-NC. Neither the total synthesis nor cellular replication properties of the N-(dA-8-yl)-6-AC or other adducts derived from 6-NC have ever been reported.
Brent Valentine Powell - University of Connecticut, 2020
Chapter 2 describes synthetic methods developed to access 6-NC modified nucleosides, phosphoramidites and oligodeoxynucleotides of the C8-dA adduct, N-(dA-8-yl)-6-AC employing an optimized Buchwald-Hartwig palladium catalyzed cross-coupling strategy, which provided a high yield of the protected N-(dA-8-yl)-6-AC adducted nucleoside. The protected N-(dA-8-yl)-6-
AC adduct was converted to the protected 3’-phosphoramidite monomer and site specifically incorporated into 12 and 15-mer oligodeoxynucleotides (ODNs) via automated solid-phase DNA synthesis. These oligodeoxynucleotides were purified by reverse phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by high resolution mass spectrometry.
Chapter 3 employs 2D NMR spectroscopy and DFT calculations to investigate the conformational preference of N-(dA-8-yl)-6-AC adducted nucleoside. We further investigated the helical structures of N-(dA-8-yl)-6-AC adduct in 12 and 15-mer oligodeoxynucleotide duplexes using circular dichroism (CD) spectroscopy. The stability of N-(dA-8-yl)-6AC adducted nucleoside was also investigated under biologically relevant conditions using UV/Vis spectroscopy and HPLC assays.
Chapter 4 describes investigation of the replication bypass efficiency of N-(dA-8-yl)-6-AC adduct in Escherichia coli and in human embryonic kidney (HEK293T) cells. The 15-mer containing N-(dA-8-yl)-6-AC modified oligodeoxynucleotide was used to construct single- stranded shuttle vectors containing a single N-(dA-8-yl)-6-AC adduct site using recombinant DNA technology. These recombinant vectors were replicated in Escherichia coli and HEK293T cells.
The roles of several translesion synthesis (TLS) replication polymerases in adduct bypass was also
Brent Valentine Powell - University of Connecticut, 2020 investigated via conducting replication experiments in isogenic cells where individual TLS DNA polymerases were depleted by the CRISPR/Cas9 genome editing method or siRNA knockdown approach.
Chapter 5 evaluates the mutagenicity and replication bypass efficiency of the ring opened formamidopyrimidine (Fapy.dG) adduct that is produced via oxidative stress in two interesting p53 mutation hotspot sequences that include codons 248 and 249. Fapy.dG modified oligodeoxynucleotides was used to construct single-stranded shuttle vectors containing a single adducted site and replicated in HEK293T cells.
Chemical Biology of 6-Nitrochrysene Induced Deoxyadenosine DNA Adduct and Formamidopyrimidine DNA Lesion: Mutagenesis and Genotoxicity in E. coli and Human Cells
Brent Valentine Powell
B.Sc., Morgan State University, 2014
A Dissertation
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy at the
University of Connecticut
2020
i
© 2020 by Brent Valentine Powell
All rights reserved.
ii Approval Page
Doctor of Philosophy Dissertation
Chemical Biology of 6-Nitrochrysene Induced Deoxyadenosine Adduct and Formamidopyrimidine DNA Lesion: Mutagenesis and Genotoxicity in E. coli and Human Cells
Presented by
Brent V. Powell
Major Advisor ______
Ashis K. Basu, Ph.D.
Associate Advisor ______
Alfredo Angeles-Boza, Ph.D.
Associate Advisor ______Amy Howell, Ph.D.
Associate Advisor ______Anwar Beshir, Ph.D.
Associate Advisor ______Mark Peczuh, Ph.D.
University of Connecticut, 2020
iii
Dedicated to my mother
Claudine Cleopatra Powell for her unyielding support and confidence in me.
iv ACKNOWLEDGEMENT
The past five and half years at UConn have been productive and have truly made me more resilient. Here, I would like to acknowledge the people who I am deeply indebted to for their advice, friendship, encouragement, and support.
Prof. Ashis Basu: I am very grateful for the opportunity to work in your research lab and for your mentorship. Over the years, you have given me insightful feedback and guidance on my work in the lab which have enabled my growth as a scientist. I have also benefitted from your immense knowledge on DNA damage and repair through the many scientific discourse we’ve shared.
Dr. Alfredo Angeles- I am grateful that you provided me with a safe space to discuss life, Boza: science and my career goals. I am also grateful that you allowed me to use instruments in your lab; your generosity greatly accelerated my research. You’ve also provided immeasurably feedback on much of my work and I am tremendously grateful.
Prof. Amy Howell: I have been motivated by your passion for chemistry and your commitment to training and mentoring students. Thank you for serving on my thesis committee and also for giving me advice before my BI internship.
Dr. Anwar Beshir: I will forever be inspired by your commitment to teaching and your decency. I’ve always enjoyed working as your TA because of your impeccable organization skills. I am very grateful to you for volunteering your time to talk about the progress of my research and helping to keep me on track.
v Prof. Mark Peczuh: I enjoyed your organic synthesis course and the many discourse we’ve had over the years. Thank you for your feedback during my general exam preparation.
Prof. Santosh Mandal: My decision to go to graduate school was entirely because of how much I appreciated working alongside you in the lab as an undergraduate student at Morgan. I wouldn’t be here today if not for you. You’ve supported me, inspired me and have always shown interest in my development as a scientist. I am tremendously grateful for your guidance, mentorship and the role you’ve played in helping to shape my career interest.
Dr. Ivan Volchkov: I am grateful for my internship opportunity at Boehringer Ingelheim and for the opportunity to work with you. I learnt so many “tricks” on how to work smarter and not harder from you. Thanks for introducing me to the wonderful world of fluorine chemistry.
Dr. Vitaliy Gorbatyuk: I am very thankful for your help with setting up my 2D NMR experiments and your willingness to assist with NMR related issues.
Dr. You-Jun Fu: I am grateful for the time you expended in training me on how to troubleshoot and develop methods on the mass spec before you left UConn. This greatly helped in accelerating my research.
Tyler Cardinal: I am so grateful for your willingness to help with troubleshooting instrumental malfunctions around the lab and for letting me borrow your tools.
Dr. Bo Reese Thank you for teaching me how to sequence plasmid DNA and welcoming me to the Center for Genome Innovation (CBI).
vi
Dr. Matthew Guberman- I am very grateful for your willingness to collaborate and for the Pfeffer: enthusiasm that you have shown while working with me.
Dr. Arindom Chaterjee: I am grateful for your friendship and to have worked with you in the Basu lab. Thank you for helping me with setting up my hood and for being such a welcoming lab mate. It was always refreshing discussing chemistry and even the stress of graduate school with you.
Spandana: I am very grateful that you allowed me to shadow you during my transition to the bio-side of the lab and for the many conversation we’ve
shared as lab mates.
Henric: Thank you for being such a considerate and helpful lab mate. I am very appreciative for your help in the lab. All the best with completing your Ph.D.
Elyse: It was really awesome to train you in the lab. I am very grateful for all the time you expended working alongside me in the lab.
Mishil: I remembered the day you asked me during organic teaching lab if you could work with me in my research lab. You’ve been a great addition to the lab. I am very grateful for all your help and company in the lab.
Ryan: It’s been a pleasure to have worked with you in the lab. I am very grateful for all the time you expended working with me on my project. All the best in your final semester.
vii Shanka and Omer I am very thankful to have met you guys at UConn Chemistry. Both of you have been great friends, coffee buddies and weekend adventure partners. My family: I am tremendously grateful for my parents Claudine and Oliver for their unconditional love, encouragement and unwavering support in every endeavor I have undertaken. To my sister, Nicki and brother Roger, I am incredibly grateful for your support and for the constant reminder of what matters the most. My nieces, Markayla and Regina and nephew Prince thanks for filling me with happiness, motivation and unsolicited facetime calls which helped to keep me sane.
Dr. Camille Richards: Finally, to my dearest love and partner Camille, I am incredibly grateful for your perpetual love, patience and unyielding support through these challenging years. You’ve always challenged me to reach for more. You’ve been a true champ, an inspiration and the best part of my little world. I look forward to our next chapter in life.
viii Table of Contents
Chapter 1 - Introduction ...... 1
1. 1 DNA Damaging Agents ...... 1
1. 2 Carcinogenicity of Nitroaromatic compounds ...... 2
1. 3 6-Nitrochrysene ...... 3
1. 4 DNA Repair Pathways and Genoprotective Networks ...... 7 1. 4. 1 Overview ...... 7 1. 4. 2 Biological Consequences of DNA Damage ...... 8 1. 4. 3 Base Excision Repair (BER) ...... 8 1. 4. 4 Nucleotide Excision Repair (NER) ...... 9
1. 5 Translesion Synthesis (TLS) ...... 10 1. 5. 1 Translesion Synthesis in Mammalian Cells ...... 11 Polymerase η ...... 12 Polymerase κ ...... 12 Polymerase ζ ...... 13 1. 5. 2 DNA Damage Response in Escherichia coli ...... 13 SOS Response ...... 13 Translesion Synthesis in Escherichia coli ...... 14
1. 6 Scope of Dissertation ...... 16
Chapter 2 - Total Synthesis of Site-Specific Oligodeoxynucleotides Containing C8-2'- Deoxyadenosine Adduct Formed by 6-Nitrochrysene ...... 19
2. Abstract ...... 20
2. 1 Introduction ...... 21 2. 1. 1 Buchwald-Hartwig Cross-Coupling Chemistry ...... 21 2. 1. 2 Synthesis of Carcinogen Modified Oligodeoxynucleotides ...... 24 Biomimetic Postoligomerization ...... 24 Phosphoramidite Chemistry Method ...... 24
2. 2 Material and Methods ...... 26
ix 2. 2. 1 Experimental Methods ...... 27 2. 2. 2 Synthesis of Site-Specifically Modified Oligodeoxynucleotides ...... 36 2. 2. 3 Purification of Site-Specifically Modified Oligodeoxynucleotides ...... 36 HPLC Purification ...... 36 PAGE Purification ...... 36
2. 3 Results and Discussion ...... 38 2. 3. 1 Buchwald-Hartwig Cross-Coupling Reaction Optimization ...... 39 2. 3. 2 Synthesis of Modified Phosphoramidite Monomer ...... 41 2. 3. 3 Synthesis of Oligodeoxynucleotides Containing N-(dA-8-yl)-6-AC Adduct ...... 42
2. 4 Conclusion ...... 44
Chapter 3 - Conformational Preference and Stability of 6-Nitrochrysene Modified 2'- Deoxyadeosine Adduct ...... 45
3. 1 Introduction ...... 47
3. 2 Materials and Methods ...... 50 3. 2. 1 Synthesis of N-(dA-8-yl)-6-AC nucleoside ...... 50 3. 2. 2 Synthesis of site-specifically modified oligodeoxynucleotides ...... 50 3. 2. 3 DFT Calculations ...... 50 3. 2. 4 Circular Dichroism Studies ...... 51 3. 2. 5 UV/Vis Spectroscopy Stability Studies ...... 51 3. 3 Results and Discussion ...... 52
3. 4 Conclusion ...... 58
Chapter 4 - Translesion Synthesis and DNA Repair of 6-Nitrochrysene-Derived C8-2′- Deoxyadenosine Adduct in E. coli and Human Cells ...... 59
4. 1 Introduction ...... 61
4. 2 Materials and Methods ...... 63 4. 2. 1 Materials ...... 63 4. 2. 2 Methods ...... 64 Agarose gel electrophoresis for single-stranded vector analysis ...... 64 Bacterial electro-competent cell preparation and SOS induction ...... 65
x Large scale preparation of single-stranded pMS2 (sspMS2) vector ...... 65 Single-Stranded pMS2 modified vector construction ...... 68 Replication of modified vector in HEK293T cells ...... 70 TLS assay in human cells ...... 70 Mutant screening by oligonucleotide hybridization ...... 71 Transformation of modified vector in E. coli and progeny analysis ...... 72
4. 3 Results and Discussion ...... 73 Mutational analyses of N-(dA-8-yl)-6-AC in E. coli ...... 74 Mutational analyses of N-(dA-8-yl)-6-AC in Human Cells ...... 78 The Role of Specific TLS Polymerases in N-(dA-8-yl)-6-AC TLS Bypass ...... 80
4. 4 Conclusion ...... 82
Chapter 5 - Mutagenicity and Genotoxicity of Fapy.dG DNA Adduct in Human Cells ...... 83
5. Abstract ...... 84
5. 1 Introduction ...... 85
5. 2 Material and Methods ...... 87 5. 2. 1 Synthesis of Fapy.dG containing oligonucleotides ...... 87 5. 2. 2. Single stranded vector construction and replication studies...... 87
5. 3 Results and Discussion ...... 88
5. 4 Conclusion ...... 90
5. 5 Future Direction ...... 90
Appendix A: Chapter 2 Supporting Information – Spectral Data ...... 107
Appendix B: Chapter 4 Supporting Information ...... 135
Appendix C: Chapter 5 Supporting Information ...... 143
xi List of Figures
Figure 1. Types of DNA damages from endogenous and exogenous agents ...... 1
Figure 2. Examples of nitroaromatic compounds ...... 2
Figure 3. Carcinogenicity scale of 6-nitrochrysene (6-NC) and reactive metabolites...... 5
Figure 4. Mechanism of nucleotide excision repair in eukaryotes. Adapted from reference 28 with permission from Cold Spring Harbor Laboratory Press...... 10
Figure 5. Mechanism of Translesion DNA synthesis (TLS) in mammalian systems. Adapted from reference 26 with permission from Cold Spring Harbor Laboratory Press...... 11
Figure 6. Induction of SOS response in E. coli. Adapted from reference 43...... 14
Figure 7. Mechanism of Translesion DNA synthesis (TLS) in E. coli. Adapted from reference 30 with permission from Cold Spring Harbor Laboratory Press...... 15
Figure 8. Site of adduction of nitroaromatic carcinogens with purine bases...... 23
Figure 9. (A) Buchwald-Hartwig cross-coupling catalytic cycle. (B) Commonly used ligands. 23
Figure 10. Synthesis cycle for solid-phase oligonucleotides synthesis...... 25
Figure 11. Mechanism of Buchwald-Hartwig cross coupling reaction of protected 8-bromo-2’- deoxyadenosine and 6-aminochrysene (6-AC) ...... 41
Figure 12. Structures of modified oligodeoxynucleotides...... 44
Figure 13. Depictions of three major conformations induced in N linked C8-aryl-dG adducts .. 47
Figure 14. Major DNA adducts derived from 6-NC via nitroreduction pathway ...... 48
Figure 15. Structures and chemical numbering of N-linked DNA adduct at the C8-site of 2′- deoxyadenosine ...... 50
Figure 16. Proton NMR spectrum of N-(dA-8-yl)-6-AC adducted nucleoside (3) ...... 52
Figure 17. ROESY NMR spectra of N-(dA-8-yl)-6-AC adduct (3) in DMSO-d6 at 300 K ...... 53
xii Figure 18. Potential energy surface scan around the deoxyribose-nucleobase bond of N-(dA-8-yl)-
6-AC adduct (3) ...... 54
Figure 19. CD curves of unmodified (red solid line) and modified (black solid line) 15-mer (Panel
A) and 12-mer (Panel B) oligodeoxynucleotides after annealing with their complementary strand.
...... 55
Figure 20. Overlay of the absorption spectra of N-(dA-8-yl)-6-AC adducted nucleoside ...... 57
Figure 21. Nucleotide excision repair of 6-NC DNA adducts in HeLa cell extracts.14 ...... 61
Figure 22. Workflow for the large-scale preparation of ssPMS2 plasmid...... 67
Figure 23. Workflow for construction of modified recombinant vector...... 68
Figure 24. Agarose gel analysis for recombinant DNA construction ...... 70
Figure 25. A general scheme for construction of the lesion-containing vector and replication in E. coli or HEK 293T cells, and analysis of the progeny ...... 73
Figure 26. (A) The bypass efficiencies in N-(dA-8-yl)-6-AC construct in E. coli (-) SOS (WT) and
(+) SOS cells. (B) The frequencies of total targeted mutations (C) induced in progeny from N-(dA-
8-yl)-6-AC construct in (-) SOS (WT) and (+) SOS E. coli cells...... 75
Figure 27. TLS bypass efficiencies of the N-(dA-8-yl)-6-AC containing construct in HEK293T cells and various TLS polymerase KO HEK 293T cells...... 77
Figure 28. Targeted and semi-targeted mutations induced in the progeny from the N-(dA-8-yl)-6-
AC construct in HEK 293T and various polymerase knockout cells...... 79
Figure 29. The types and frequencies of targeted mutations induced in the progeny from the N-
(dA-8-yl)-6-AC construct in HEK 293T cells and various polymerase knockout HEK293T cells.
...... 80
Figure 30. Oligonucleotide sequences from the p53 hotspots ...... 86
xiii Figure 31. Progeny plasmid processing via oligonucleotide hybridization and DNA sequencing
...... 88
Figure 32. (A) The bypass efficiencies of Fapy.dG constructs (G1 and G3) in HEK293T cells. (B)
The frequencies of total targeted mutations induced in progeny from Fapy.dG construct in dG1-
Fapy and dG3-Fapy...... 89
List of Schemes
Scheme 1. Metabolic activation of 6-nitrochrysene via nitroreduction and ring oxidation ...... 3
Scheme 2. Generation of nitrenium ion as ultimate carcinogen ...... 4
Scheme 3. Buchwald-Hartwig cross coupling reaction overview ...... 22
Scheme 4. Examples of arylmine C8-dA Buchwald-Hartwig cross coupling reactions ...... Error!
Bookmark not defined.
Scheme 5.Preparation of Fully Protected Br-dA...... 38
Scheme 6. Synthesis of Corresponding 3’-Phosphoramidite 10 ...... 42
Scheme 7. Modified oligodeoxynucleotides deprotection and purification ...... 43
Scheme 8. Postulated hydroxyl radical mediated pathway to Fapy.dG and 8-oxo-dG ...... 85
List of Tables
Table 1. Mammary tumor induction by 6-NC and reactive metabolites ...... 6
Table 2. Optimization of Buchwald-Hartwig cross coupling reaction...... 40
Table 3. HRMS-ES mass for modified and unmodified (control) oligodeoxynucleotides ...... 43
xiv
List of Abbreviation
6-AC 6-aminochrysene
6-NC 6-nitrochrysene
N-OH-6-AC. N-hydroxy-6-aminochrysene
2-deoxyribose 2-deoxy-D-erythro-pentose
8-Me-dG 8-methyl-2’-deoxyguanosine
8-Oxo-dG 7,8-dihydro-8-oxo-2’-deoxyguanosine
ATP Adenosine triphosphate
ACN Acetonitrile
BER Base excision repair
COSY Correlation Spectroscopy
DNA Deoxyribonucleic acid
DDR DNA damage response
DFT Density functional theory dsDNA Double-stranded DNA
E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid
EtOH ethanol
ESI Electron spray ionization
HEK293T cells Human embryonic kidney cells
HMBC Heteronuclear multiple bond correlation
HPLC High performance Liquid Chromatography
xv KD Knock down
KO Knock out
LCMS Liquid chromatography–mass spectrometry
NER Nucleotide excision repair
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate-buffered saline
Pols Polymerases
Pol η Polymerase η (eta)
Pol κ Polymerase κ (kappa)
Pol ι Polymerase ι (iota)
Pol ζ Polymerase ζ (zeta)
RNA Ribonucleic acid
ROS Reactive oxygen species ssDNA single-stranded DNA
TFA triflouroacetic acid
TLS Translesion DNA synthesis
UV Ultraviolet
xvi
Chapter 1 Introduction
1
Chapter 1 – Introduction
1. 1 DNA Damaging Agents
DNA is the repository of all the genetic information in living cells. Thus, its structural integrity and stability are essential to life.1,2 However, DNA is not an inert entity, and therefore may become damaged by reactive chemical modalities that are generated in cells or that may enter cells from exogenous sources. Exposure to a variety of DNA damaging agents can lead to an estimated rate of up to 100,000 lesions per cell per day.2 This status quo requires a constant excision and replacement of damaged residues by DNA repair machinery in order to counteract potentially genotoxic and mutagenic outcomes. The reaction of genotoxic agents with DNA result in a plethora of lesions, commonly referred to as adducts. Endogenous agents include chemicals generated from normal cellular processes such as respiration and lipid peroxidation.3,4 These processes generate reactive oxygen species (ROS) that may damage DNA. Genomic DNA may also be assaulted by exogenous or environmental agents.5 The main perpetrators are ionizing radiation and genotoxic chemicals that may enter cells. Genotoxic chemicals are often metabolically activated in cells to form reactive metabolites that engage DNA in chemical reactions resulting in covalent linkages to specific sites of the four nucleotides (dNTPs) of DNA to generate DNA adducts or lesions.6–8
DNA-Protein Cross Links (DPC) Reactive Oxygen Species (ROS) DNA Damaging Drugs Abasic Sites Intra-Strand Cross links
Alkyl Protein O Double Strand Breaks NH
U
Base mismatch Base Insertion Single Strand Breaks UV light Inter-Strand Links Genetoxic Chemicals
Figure 1. Types of DNA damages from endogenous and exogenous agents
1 1. 2 Carcinogenicity of Nitroaromatic compounds
DNA damage by genotoxic chemicals can become important in the development of
7,9 biologically relevant events and human cancers. Many nitroaromatic hydrocarbons (NO2-PAH) are known to induce tumors in experimental animals.7,10,11 Nitroaromatic compounds are environmental pollutants that are present in many mixtures such as cigarette smoke, coal fly ash, diesel exhaust and have also been detected in burnt grill food. Notably, drugs containing nitroaromatic motifs have also been marketed worldwide. Thus, human exposure to nitroaromatic compounds is inevitable.12
NO2
NO2
NO2 NO2 O
6-Nitrochrysesne (6-NC) 3-Nitrobenzanthrone (3-NBA) 3-Nitrofluoranthene 2-Nitrofluorene (2-NF)
O COOH O2N NO2 O NO2
N NO2 OCH3 O
Aristolochic acid (AA) 3-Nitropyrene 4-Nitroquinoline-1-oxide (4-NQO) 4-Nitrobiphenyl (4-NBP) Figure 2. Examples of nitroaromatic compounds
In the postindustrial era, food appears to be the most significant source of NO2-PAH exposure. In addition, simultaneous exposure to PAH and nitrogen oxide gas appears to lead to endogenous
8,12 formation of NO2-PAH.
In cells, most nitroaromatic compounds are metabolically activated to reactive modalities via nitroreduction. However, oxidative pathways also contribute to the metabolism of many of these compounds. Notably, significant difference in the enzymes responsible for nitroreduction and
2 oxidation has been observed in different organisms. In humans, xanthine oxidase and microsomal
NADPH-cytochrome c have been identified as the enzymes responsible for nitroreduction, while the cytochrome P450 family of enzymes is predominantly responsible for the oxidative metabolism of these compounds.9,12,13
1. 3 6-Nitrochrysene
6-Nitrochrysene (6-NC) belongs to the family of NO2-PAH above. 6-NC is a potent mutagen in bacteria. It is an exceptionally potent carcinogen in newborn mice, and it induces mammary carcinoma in rats. The genotoxicity of 6-NC is derived from its ability to chemically react with 2’- deoxyguanosine (dG) and 2’-deoxyadenosine (dA) in DNA to generate DNA-carcinogen adducts
(Scheme 1).13–15
OH OH
Ring oxidation Nitroreduction
Nitroreduction P450 enzymes NHOH NO2 NHOH 6-NC
DNA
DNA Adducts O
N NH N O N N HO H N O NH NH2 N H N N NH2 OH HO O 5-(dG-N2-yl)-6-AC (2) OH
N-(dG-8-yl)-6-AC (1)
NH2 O N N N NH N N H H N N N N HO O HO O
OH OH
N-(dA-8-yl)-6-AC (3) N-(dI-8-yl)-6-AC (3’)
Scheme 1. Metabolic activation of 6-nitrochrysene via nitroreduction and ring oxidation
3 Several in vivo studies in mice and rats have demonstrated that 6-NC can be activated by two major pathways. These include a nitroreduction and a nitroreduction-ring oxidation tethered pathway (Scheme 1).13 The nitroreduction pathways involves a simple nitroreduction of 6-NC to form the corresponding N-hydroxy-6-aminochrysene (N-OH-6-AC). This N-OH-6-AC electrophilic species reacts with the nucleophilic C8-site of purines bases in dG and dA to generate
DNA adducts, N-(dG-8-yl)-6-AC (1), and N-(dA-8-yl)-6-AC (3). Adduct 3 is believed to give rise to the corresponding 2'-deoxyinosine adduct N-(dI-8-yl)-6-AC (3’), but whether the process of deamination is enzymatic or non-enzymatic has never been established. N-OH-6-AC electrophilic species also reacts at the N2 site of dG to form 5-(dG-N2-yl)-6-AC (2). Additional adducts are formed by a combination of ring oxidation and nitroreduction (Scheme 1).13,14,16
Nitroreduction O-Sulfotransferase P450 enzymes O NO NHOH N 2 H O S O Proximate carcinogen O
Bond fission
H N N H H Ultimate carcinogen
C8-Site of dA or dG N2-dG
O N NH H NH HN N N 2 H N dR 2 N N H H O N H N N dR
O
NH2 N NH N N N N N H HN N N dR
dR H2N
N-(dA-8-yl)-6-AC 5-(dG-N2-yl)-6-AC
Scheme 2. Generation of nitrenium ion as ultimate carcinogen
4 As with many nitroaromatic compounds, metabolic activation via nitroreduction of
6-nitrochrysene to its N-hydroxy-6-aminochrysene, N-OH-6-AC (Scheme 1) is a proximate carcinogen which can react with nucleobases in cellular DNA to induce DNA adducts. However, it is believed that the N-hydroxy-6-aminochrysene species is further activated by O-esterification or N,O-diesterification enabled by cellular enzymes such as the acetyl- and sulfotransferases
(Scheme 2). The sulfonylhydroxyamines (Scheme 2) spontaneously undergo heterolytic N-O fission to form highly reactive nitrenium ions, which is the ultimate carcinogen species that react with DNA molecules to form the corresponding DNA adducts.17,18
Intramammary administration of 6-NC metabolites resulted in the formation of mammary tumors at the site of injection (Table 1). The carcinogenic potency of 6-NC and its nitroreduction and ring-oxidation metabolites toward the mammary gland of rats was ranked in the following order: 6-NC > 1,2-DHD-6-NC > 6-AC > 6-NCDE > 1,2-DHD-6-AC (Figure 3). Taken together, these results suggest that metabolites derived from both ring-oxidation and nitroreduction contribute to the overall carcinogenicity of 6-NC in the rat mammary gland. 7,10
OH OH OH OH OH OH
O
NH2 NO2 NH2 NO2 NO2
1,2-DHD-6-AC 6-NCDE 6-AC 1,2-DHD-6-NC 6-NC
Increasing Carcinogenicity Figure 3. Carcinogenicity scale of 6-nitrochrysene (6-NC) and reactive metabolites.
5
Compounds Tumor Incidence (%) 1. 6-NC 95.8 2. 1,2-DHD-6-NC 37.5 3. 6-Ac 48 4. 6-NCDE 16 5. 1,2-DHD-6AC 28 6. Control DMSO 0 Table 1. Mammary tumor induction by 6-NC and reactive metabolites.10
6-Nitrochrysene is noticeably less abundant than other NO2-PAHs in the environment.
10 However, it is the most potent NO2-PAH compound ever tested in the newborn mouse assay.
6-NC is much more active than chrysene, and any of the other mononitro-chrysene isomers and benzo[a]pyrene (B[a]P). It has been shown that 6-NC is a powerful mammary carcinogen in rats.
In this study it was more potent than the extensively investigated ultimate carcinogen, the bay region diol epoxide of B[a]P. 6-NC is not only carcinogenic in mammary gland, it is tumorigenic in the colon of rats, and the lung and skin of mice.7,9,13 It is known that 6-NC can become metabolically activated in human liver and lung to form genotoxic metabolites.7,15
DNA lesions derived from 6-NC can play important roles in the development of human cancer if they are not repaired via cellular repair pathways such as nucleotide excision repair prior to DNA replication. These lesions can induce mutations in crucial sequences of cancer genes.
Mutations in an oncogene, a tumor-suppressor gene such as p53, or a gene that controls the cell cycle can lead to uncontrolled cell growth, resulting in tumorigenesis.19,20 A large fraction of mutations in most human cancers involves mutations in the G:C base pairs of critical genes like p53 and a relatively smaller fraction of mutations occurs at the A:T base pairs.20–22 Indeed,
6 carcinogen-induced mutational signatures also predominantly involve G:C base pairs.23 Even so, mutations at A:T base pairs occur. An example of a nitroaromatic compound derived mutations at the A:T base pair include aristolochic acid (AA), which forms N6-dA adducts. Mutational spectra of AA is dominated by A:T→T:A transversions in the p53 gene in urothelial tumors.22,24
Recently, an in vitro repair study by El-Bayoumy and coworkers showed that the efficiency of nucleotide excision repair (NER) of the C8-dA adduct, N-(dA-8-yl)-6-AC (3) is ~8 times lower than that of the C8-dG adduct, N-(dG-8-yl)-6-AC (1). In fact, N-(dA-8-yl)-6-AC was estimated to be more resistant to NER than all other adducts formed by 6-NC and benzo[a]pyrene diol epoxide.14
1. 4 DNA Repair Pathways and Genoprotective Networks
1. 4. 1 Overview
DNA damages challenge the integrity of the genome and can stall replication and transcription machinery thereby inducing mutations and the onset of diseases. However, the process of evolution has equipped cells with the tools to identify and rectify DNA damages prior to DNA replication. In most cases, assaults on DNA are identified and cells elicit complex repair mechanisms that rectify damages. This mechanism is known as DNA damage response (DDR) and involves a series of coordinating enzymes, which removes DNA damages and reassemble
DNA fragments.1 Thus, deleterious and mutagenic consequences of DNA damages are either minimized or completely eliminated. DNA damage signaling and cellular checkpoints coordinate
DNA repair with cell-cycle transitions and can effectively halt the growth of a cell with damaged
DNA until its DNA is repaired. The types of DNA lesion that results from a DNA damaging event controls the repair pathway elicited by cell’s repair machinery. DNA damages that results in
7 nucleobase alterations are usually removed by error-free pathways such as base excision repair
(BER) and nucleotide excision repair (NER).3,4
1. 4. 2 Biological Consequences of DNA Damage
Despite cell’s robust and precise ability to identify and repair damaged DNA, some damages to DNA may escape cell’s repair machinery, particularly when the damages are induced at high frequency. Replication of damaged DNA that eludes cellular defense mechanisms poses a challenge to DNA replication.1,2 First, replication of unrepaired lesions cause mutations, which can result in genetic diseases. Second, DNA adducts can block replicative DNA polymerases, which are cellular workhorses responsible for copying DNA with high fidelity during replication.
High fidelity replicative polymerases can fit only one templating nucleotide in their active site.25,26
This favors accurate Watson-Crick-Franklin base pairing with incoming nucleotide of the newly made complementary strand of DNA. RNA and DNA polymerases blockages at replication forks can cause genome instability by chromosomal aberrations and can lead directly or indirectly to
DNA strand breaks, which tend to be lethal in most cells.26,27
The detrimental biological consequences that DNA damage can present cells with is evident by the large commitment of genome protection expended by cell’s genoprotective networks including electrophile scavengers, recombination complexes that permit DNA lesion tolerance, specialized polymerases that enable lesion bypass and a large arsenal of DNA repair proteins.3 The inability of this protective network to effectively protect the genome can result in the onset of diseases.
1. 4. 3 Base Excision Repair (BER)
In human cells, base modifications that’s are caused by endogenous agents such as reactive oxygen species (ROS) are generally removed by the base excision repair pathway.1–3 BER is
8 initiated by DNA glycosylases that hydrolytically cleave the base–deoxyribose glycosyl bond of a damaged nucleotide residue. This results in formation of an abasic site. The coordinated activities of AP endonuclease (APE1) and polymerase β then insert the correct base and the nicks are sealed by XRCC1-ligase 3 complex.28,29 The insertion of the appropriate base by polymerase β, can be accomplished in two different mechanisms, short-patch and/or long-patch repair. In short-patch repair DNA pol β performs a one nucleotide gap filling reaction and removes the AP site via lyase activity.29
1. 4. 4 Nucleotide Excision Repair (NER)
The nucleotide excision repair pathway is mainly involved in the direct repair of bulky DNA lesions such as those induced by nitroaromatic chemical carcinogens, UV radiation and DNA damaging drugs. These distorting lesions thermodynamically destabilize DNA double helix and the enzymes involve in global NER utilize these perturbations to recognize and repair these lesions
(Figure 4).30
As depicted in Figure 4, distorting lesions are directly recognized by the enzyme XPC-
RAD23B, which binds the nondamaged strand opposite the lesion. TFIIH interacts with XPC-
RAD23B through protein-protein interaction and effectively forces the DNA helix wide open with its XPB subunit allowing XPD to slowly moves along DNA until its stalls at the damage site. This stalling of XPD at the lesion allows for the formation of the pre-incision complex by recruitment of other enzymes such as XPA, RPA, and XPG which cooperatively makes an incision 5’ to the lesion. DNA repair synthesis is then initiated by Pol δ and Pol κ or Pol ε and associated factors, followed by 3’ incision by XPG. The newly synthesized strand is stitched to the undamaged strand by DNA ligase which seals the nick and completes the process.30,31
9
Figure 4. Mechanism of nucleotide excision repair in eukaryotes. Adapted from reference 28 with permission from Cold Spring Harbor Laboratory Press.
1. 5 Translesion Synthesis (TLS)
In order to evade the lethal consequences of excessive DNA damage, cells have developed a specialized mechanism known as translesion DNA synthesis (TLS), a pathway that is able to tolerate DNA damages in the genome by briefly replacing high fidelity replicative DNA polymerases with TLS polymerases.27,32 These specialized enzymes can bypass DNA lesions by inserting the correct or incorrect base opposites the lesion site.27,32,33 However, TLS polymerases are low fidelity enzymes which makes TLS an inherently error-prone process. Unlike replicative polymerases with tight binding sites, translesion polymerases have a solvent-exposed active site that is spacious enough to accommodate bulky DNA lesions, while enabling low fidelity and potential mutagenic replication.25
10 1. 5. 1 Translesion Synthesis in Mammalian Cells When replicative polymerases such as eukaryotic Pol ε (Figure 5) stalls at the replication fork upon encountering a DNA lesion site, TLS polymerases are recruited to the site. TLS polymerases are recruited through protein-protein interactions with the replicative sliding processivity clamp (yellow).26,27
Figure 5. Mechanism of Translesion DNA synthesis (TLS) in mammalian systems. Adapted from reference 24 with permission from Cold Spring Harbor Laboratory Press.26
This is known as proliferating cell nuclear antigen (PCNA) in eukaryotes. In eukaryotic systems stalling of replicative polymerase causes the proliferating cell nuclear antigen (PCNA) to becomes ubiquitinated by the protein ubiquitin.26 This effectively signals the release of replicative polymerase from the sliding clamp of the replisome and recruit TLS polymerases to bind to the
11 replication fork. These polymerases include Y family polymerases such as pol η, pol κ, pol ι, Rev1 and the B family polymerase Pol ζ.26,27,33 TLS polymerases insert a correct or an incorrect base opposite the lesion. Translesion DNA synthesis is subsequently extended in the 5’ to 3’ direction past the lesion by the same TLS polymerase or a second TLS polymerase.26,33 After synthesizing a short sequence of DNA, all TLS polymerases are dissociated from the replication fork and replaced with high fidelity replicative polymerase to resume DNA synthesis.
Polymerase η
Polymerase η (Pol η) is a Y-family polymerase and is the most extensively studied TLS polymerase. Pol η bypasses UV-induced thymine-thymine cyclobutane pyrimidine dimers (CPD) with high efficiency and high fidelity.34 In fact, Pol η is capable of bypassing these lesions with higher accuracy than even undamaged DNA. In humans, the presence of mutations in the POLH, gene responsible for the expression of Pol η, is known to result in the variant form of xeroderma pigmentosum (XP)33,35, a disease associated with an increased incidence of skin cancer. Pol η has been labeled as CPD bypass polymerase due to its ability to bypass the CPD lesions with high fidelity. However, Pol η have been shown to play important roles in mutagenic and error free bypass many other lesions, such as to bypass 8-oxodG,36 O6-MedG,37 benzo[a]pyrene-N2-dG,37 and DNA lesions derived from cisplatin.38
Polymerase κ
Polymerase κ (Pol κ) is found in all domains of life and is the most highly represented and strongly conserved of all the TLS Y-family polymerases.26 Pol κ is considered to be the most accurate y-family polymerase on processing undamaged DNA.27,34 Notably, Pol κ has the unique
12 capability in accurately bypassing various dG lesions, including C8-guanyl,39 N2-furfuryl-dG40 and 8-oxo- guanine.33
Polymerase ζ
Unlike most mammalian TLS polymerases which are low fidelity Y-family polymerases, polymerase ζ (Pol ζ) belongs to B-family class of polymerases. Notably, other B-family polymerases includes high fidelity replicative polymerases such as Pol delta (δ), epsilon (ε) and alpha (α).26,33,34 However, unlike these replicative polymerases, Pol ζ is error-prone and lacks 3’-
5’ exonuclease activity which makes it categorically a TLS polymerase. Pol ζ is a herterodimer composed of the Rev3 catalytic subunit and the Rev7 accessory subunit. Many researchers have shown that Pol ζ participates in a variety of error-free and error-prone bypass of many DNA lesions.41,42
1. 5. 2 DNA Damage Response in Escherichia coli
SOS Response
In E. coli DNA repair is regulated by the expression of a network of genes which are referred to as the SOS response. Induction of the SOS response involves more than forty independent SOS genes, most of which are responsible for the expression of proteins responsible for protection,
DNA repair and TLS lesion bypass.43,44 Under normal growth conditions the SOS genes are expressed at a basal level. These levels increase significantly when the SOS genes box is turned on in response to genome damage. Thus, in bacterial systems the expression of TLS polymerases is regulated by SOS response.45
The expression of genes in the SOS box is regulated by the LexA protein. Under normal cellular conditions, LexA dimer is bound to the promoter region of the SOS box thereby inhibiting
13 gene expression (Figure 6).45,39 Upon encountering a replication blockage at the replication fork during DNA replication the RecA protein is activated to become a coprotease by formation of a filament of RecA on single-stranded DNA (ssDNA). RecA is a protease and facilitates the self- cleavage reaction of LexA. As LexA repressor levels decrease, the SOS genes are expressed and induces DNA repair mechanisms, tolerance of DNA damage, and induced delay of the cell cycle.44,45
Figure 6. Induction of SOS response in E. coli. Adapted from reference 43.
Translesion Synthesis in Escherichia coli
In E. coli, TLS low fidelity polymerases are Pol II, Pol IV and Pol V. When replicative polymerase Pol III (Figure 7) stalls at the replication fork upon encountering a site of a DNA lesion, TLS polymerases are recruited to the site.26,32 TLS polymerases are recruited through interactions with the replicative sliding processivity clamp (yellow). This is known as β-Clamp in
E. coli. This signals the release of replicative Pol III from the sliding clamp of the replisome and
14 recruit TLS polymerases to bind to the replication fork. These polymerases include low fidelity polymerases are Pol II, Pol IV and Pol V. TLS polymerases insert a correct or an incorrect base opposite the lesion. Translesion DNA synthesis is subsequently extended past the lesion by the same TLS polymerase or a second TLS polymerase. After synthesizing a short sequence of DNA, all TLS polymerases are dissociated from the replication fork and replaced with high fidelity replicative polymerase to resume DNA synthesis.26
Figure 7. Mechanism of Translesion DNA synthesis (TLS) in E. coli. Adapted from reference 30 with permission from Cold Spring Harbor Laboratory Press.
15 1. 6 Scope of Dissertation
It is now clear that carcinogen-DNA adducts can interfere with DNA structure and affect the accuracy of DNA replication leading to heritable genetic changes and cancer. The overarching goal of my thesis research is to investigate the mechanisms of mutagenesis and DNA repair induced by DNA lesions in cells. The multi-faceted nature of this goal requires tools in organic synthesis, analytical chemistry and molecular and cell Biology.
Chapter 2 of this thesis focus on the synthesis of site-specifically modified oligodeoxynucleotides containing C8-deoxyadenosine DNA adduct derived from 6-nitrochrysene
(6-NC) modification. This lesion derives from the nitroreduction 6-NC to its reactive metabolite which subsequently reacts at the C8 site of 2’deoxyadenosine in DNA to generate N-(dA-8-yl)-6-
AC DNA adduct. This adduct is referred to as N-(dA-8-yl)-6-AC throughout this thesis. 6-NC is the most potent carcinogen ever tested in newborn mouse assay and is a potent mutagen in bacteria. DNA lesions resulting from 6-NC modifications can inhibit DNA replication and are likely to induce mutations if they are not removed by cellular defense pathways prior to DNA replication. The N-(dA-8-yl)-6-AC DNA adduct is particularly interesting; it has been showed to be poorly repaired in nucleotide excision repair assay in HeLa cells extract. Using phosphoramidite chemistry and solid phase DNA synthesis, I have successfully synthesized this N-(dA-8-yl)-6-AC adduct. The key step in the synthesis of N-(dA-8-yl)-6-AC DNA adduct is a palladium-catalyzed
Buchwald-Hartwig cross coupling reaction wherein carcinogenic 6-aminochrysene is coupled at the C8 site of protected bromo-dA. The 6-NC modified adduct, N-(dA-8-yl)-6-AC was converted to its corresponding 3’-phosphoramidite precursor and used to synthesize 12-mer and 15-mer oligodeoxynucleotides. These oligodeoxynucleotides were designed to mimic parts of the p53 tumor suppressor gene with biologically relevant mutation hotspots. N-(dA-8-yl)-6-AC adduct
16 containing oligodeoxynucleotides were purified by reversed phase HPLC and denaturing polyacrylamide gel electrophoresis and characterized by high resolution mass spectrometry.
Chapter 3 address the biophysical characterization of the 2’-deoxyadenosine DNA adduct,
N-(dA-8-yl)-6-AC. In this section, 2D NMR spectroscopy and DFT calculations were used to investigate the conformational preference of N-(dA-8-yl)-6-AC adducted nucleoside. We further investigated the helical structure of N-(dA-8-yl)-6-AC adduct in the 12 and 15-mer oligodeoxynucleotide duplexes using circular dichroism (CD) spectroscopy. The stability of
N-(dA-8-yl)-6AC adducted nucleoside was also investigated under biologically relevant conditions using UV/Vis spectroscopy and HPLC.
The focus of chapter 4 is to investigate the replicative bypass of N-(dA-8-yl)-6-AC adduct in E. coli and human embryonic kidney cells, HEK293T cells. The 15-mer oligodeoxynucleotide
(5’-GCCCTCAA*CAAGATG-3’) where A* = N-(dA-8-yl)-6-AC adduct was used to construct a single-stranded shuttle vector containing a single adducted site using recombinant DNA technology and replicated in cells. The DNA sequence of the 15-mer was chosen from TP53 gene codon 129-133, because crops contaminated with another nitroaromatic carcinogen aristolochic acid (AA) causes A→T mutation in codon 131 in patients with urothelial tumors. In Escherichia coli, viability of N-(dA-8-yl)-6-AC modified vector was ~60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. N-(dA-8-yl)-6-AC DNA adduct is mutagenic in E. coli. The major type of targeted mutation is A*→G in both uninduced and SOS-induced cells.
The roles of translesion synthesis DNA polymerases in bypassing the 6NC modified C8-2'- deoxyadenosine adduct, N-(dA-8-yl)-6-AC in HEK293T were also investigated in chapter 4.
17 Replication of plasmid containing a single site-specific N-(dA-8-yl)-6-AC adduct in HEK293T provided ~12 % progeny plasmid with mutations largely A*→T transversions. A 4-fold decrease in mutation frequency was observed when the plasmid is replicated in polymerases κ or ζ deficient cells. Similar experiments in polymerase η KO cells showed a 1.5-fold increase in mutation frequency. We also noted that plasmid replication in polymerases ζ KO and κ siRNA KD cells resulted in a 10-fold decrease in mutation frequency. Taken together, these results suggest that polymerases κ and ζ are cooperatively involved in the error-prone TLS of N-(dA-8-yl)-6-AC, while pol η is performs error-free bypass.
Chapter 5 evaluates the mutagenicity and replication bypass efficiency of the ring opened formamidopyrimidine (Fapy.dG) adduct that is produced via oxidative stress. DNA sequences:
5’-AAC CG*G AGG CCC-3 and 5’-AAC CGG AG*G CCC-3 with codon 248 and 249
(underlined) respectively of the tumor suppressor gene p53 was incorporated into a plasmid and replicated in human embryonic kidney 293T cells, which showed that Fapy.dG DNA adduct is mutagenic inducing largely G*→T transversions. Furthermore Fapy.dG adduct in CG*G sequence context (codon 248) induce mutations at ~50% greater frequency than AG*G in codon 249.
18
Chapter 2
Total Synthesis of Site-Specific Oligodeoxynucleotides Containing C8-2'-Deoxyadenosine Adduct Formed by 6-Nitrochrysene
†Powell, B.V.; Basu, A.K. “6-Nitrochrysene-Derived C8-2’-Deoxyadenosine Adduct: Synthesis of Site-Specific Oligodeoxynucleotides and Mutagenicity in Escherichia coli. 2020 Chem Res Toxicol [In Press]
19 2. Abstract
The environmental pollutant, 6-nitrochrysene (6-NC) is a potent mutagen in bacteria and it induces mammary carcinoma in rats. It is an exceptionally potent carcinogen in newborn mice.
6-NC is metabolically activated by two pathways, which include only nitroreduction and both nitroreduction and ring oxidation. The nitroreduction pathway generates dG-C8, dG-N2, and the dA-C8 adduct, N-(dA-8-yl)-6-AC. Here, we report the synthesis of the phosphoramidite monomer of the C8-dA adduct of 6-NC from dA in 10 steps and its incorporation into oligodeoxynucleotides.
The key step in the synthesis of oligodeoxynucleotides containing N-(dA-8-yl)-6-AC was palladium-catalyzed Buchwald-Hartwig cross coupling chemistry, which was optimized to achieved in 85% yield. The protected N-(dA-8-yl)-6-AC derivative was deprotected and then converted to appropriately protected phosphoramidite monomer for solid-phase DNA synthesis.
The resulting phosphoramidite was used to synthesize a 12-mer and a 15-mer oligonucleotide. The adduct-containing oligodeoxynucleotides were purified by reversed phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by mass spectrometry.
20 2. 1 Introduction
2. 1. 1 Buchwald-Hartwig Cross-Coupling Chemistry
NH2 NH2
N N Ar-NH2 N N Br Pd (dba) Ar N N 2 3 N N BINAP H N RO O RO O NaOt-Bu OR OR toluene OR OR 85 - 110 º C R = tertbutyldimethylsilyl 8 - 24 h
NH2 NH2 H2N NH NH2 Ar-NH2 = 2
84% 56% 60% 42% Schoffers, E. et. al. Org.Lett.,Vol.3, No. 26 2001, 4221
NH2 NH2 Ar-NH N N 2 N N Br Ar N Pd (dba) N N 2 3 H N N BINAP RO O RO O Cs2CO3 OR 1, 2 DME OR 95 º C R = tertbutyldimethylsilyl 24 - 48 h
NH2 NH2
NH Ar-NH = 2 2 H2N NH2
77% 73% 90% 82% 91%
Szombati, Z. et. al. ChemBioChem, 2012, 13, 700
DMT HN DMT HN NH N N N 2 N Br Pd2(dba)3 Ar N N xanphos H N N N + RO RO O O Cs2CO3 O toluene, 100 º C OR 3 h OR 80% R = tertbutyldimethylsilyl Ar-NH2
Takamura-Enya, T. et.al Chem.Asian J. 2007, 2, 1174 Scheme 3. Examples of arylamine C8-dA Buchwald-Hartwig cross coupling reactions
21 The Buchwald-Hartwig palladium catalyzed C-N bond formation strategy has been successfully applied by many researchers to synthesize various DNA adducts (Scheme 3).46–50
This strategy involves protection of the reactive functionalities on the nucleoside substrate and any necessary functionalization that facilitates smooth coupling. The Buchwald–Hartwig reaction is a cross-coupling reaction where C-N bond of an arylamines or heteroarylamines is formed by the reaction of an aryl halide or triflate with a primary or secondary amine in the presence of a palladium (0) catalyst and a base (Scheme 4).51–53
R2 N X Pd (0) catalyst R1 R2 R1 + N Ligand, Base H R R
X = Cl, Br, I, OTf Amine = 1º, 2º and R1/R2 = aromatic or aliphatic substituent
Scheme 4. Buchwald-Hartwig cross coupling reaction overview
Typically for carcinogen-DNA coupling, C-N bond connecting the NO2-PAHs carcinogen and the nucleoside base is installed via a Buchwald-Hartwig coupling reaction. The point of adduction of the purine bases varies. Nitroaromatic carcinogens-DNA attachments can occur at the C8 and N2 site deoxyguanosine (dG) or at the C8 and N6 deoxyadenosine (dA) as shown in
Figure 8.
22 carcinogen O O NH2 HN N N N N N N carcinogen NH NH carcinogen N N N N NH2 N N NH N N HO HO HO O HO O O O
OH OH OH OH carcinogen
C8-dG N2-dG C8-dA site N6-dA Figure 8. Site of adduction of nitroaromatic carcinogens with purine bases.
The Buchwald-Hartwig cross coupling reaction begins with oxidative addition of the aryl halide or triflate species to the palladium which increases the oxidation state of Pd from 0 to +2.
This is followed by coordination of the amine to the palladium. An attack of amine on the palladium (II) complex kicks out the halide as a leaving group and react with a strong base in solution. Reductive elimination then produces the final aryl amine product and regenerates the catalyst (Figure 9).52,53
Pd(BINAP)2
Ar X Ar NR1R2
(BINAP)Pd [0] Ph A B P Ph P Ph OCH3 iPr Ph iPr rac-BINAP H3CO P iPr
[II] [II] R R N BINAP X BINAP 2 1 Pd Pd tBu P tBu Ar Ar BrettPhos
TrixiePhos
NaX + tBuOH NaOtBu + R1 R N 2 H Figure 9. (A) Buchwald-Hartwig cross-coupling catalytic cycle. (B) Commonly used ligands.
23 The most common chelating ligand used in nitroaromatic carcinogen-nucleobase Buchwald-
Hartwig cross coupling reactions is 2,2’-bis(diphenylphosphino)-1,1’-binapthyl (BINAP).54
However, other ligands have also been used successfully. Generally, these ligands are bulky electron-rich dialkylbiaryl phospines.55,56
2. 1. 2 Synthesis of Carcinogen Modified Oligodeoxynucleotides
Biomimetic Postoligomerization
To investigate mutation outcomes in cells carcinogen modified oligodeoxynucleotides are often synthesized via a biomimetic postoligomerization stategy or using phosphoramidite chemistry. In a postoligomerization approach an unmodified oligodeoxynucleotide is directly reacted with a carcinogen or its reactive metabolite. The resulting oligomer is then purified and characterized. The recovery of these reactions is inherently low yielding and only a single reactive base is accommodated in the starting unmodified oligodeoxynucleotide. Multiple reactive bases in the unmodified oligo substrate increases the number of possible products exponentially which makes purification difficult and further decreases the yields.13,14,50
Phosphoramidite Chemistry Method
The synthesis of site-specifically carcinogen modified oligodeoxynucleotides utilizing phosphoramidite chemistry and solid phase DNA synthesis is currently the gold standard for synthesizing modified oligodeoxynucletides.57 Phosphoramidite chemistry, developed in the
1980s and later enhanced with solid-phase supports and automation, is the method of choice for
DNA oligonucleotide synthesis.58–60 Unlike biosynthesis, which proceeds in 5' → 3' direction, solid phase DNA synthesis proceeds in the 3' → 5' direction according to the steps outlined in Figure 10. The synthesis cycle begins with detritylation, the 5'-DMT protecting group is removed from the first, solid-support-linked nucleoside. In step 2, coupling, the free 5'-OH of the
24 first, solid-support-linked nucleoside attacks the phosphorus of the incoming second nucleoside, displacing its diisopropylamino group. This is the step in which modified phosphoramidites can be introduced. In step 3, oxidation, the resulting phosphite triester is unstable and is converted to a stable phosphate triester, this allows the next cycle to proceed to step 1. However, before moving to the next cycle, unreacted nucleosides with 5'-OH on the solid support are acetylated or capped, thereby preventing elongation of sequences with deletion mutations.
1. Deblocking Base 1 Base 1 DMT O O HO O Base 2 O O DMT O O Linker Linker O P NC O N
4. Capping 2. Coupling Base 2
DMT O O Base 2 O DMT O NC O O P O Base 1 O O O NC O P Base 1 O O O Linker
O Linker 3. Oxidation
Figure 10. Synthesis cycle for solid-phase oligonucleotides synthesis.
25 2. 2 Material and Methods
All reagents were purchased from commercial chemical suppliers and used without further purification unless otherwise noted. All reactions were performed in round bottomed flasks sealed with rubber septa, under an atmosphere of nitrogen, and stirred with a TeflonTM-coated magnetic stir bar unless otherwise noted. Temperatures above 23 °C were controlled by a temperature modulator. Pre-dried tetrahydrofuran (THF), benzene, toluene, acetonitrile (MeCN), methanol
(MeOH), and triethylamine (Et3N), were degassed with argon for 60 min. Reactions were monitored by thin layer chromatography (TLC) using J.T Baker 2.5 x 7.5 cm silica gel flexible
TLC sheets and visualized using UV (254 nm). Volatile solvents were removed using a rotary evaporator under reduced pressure. Silica gel chromatography was performed using Sorbent
Technologies 60 A, 230 x 400 mesh silica gel (40-63 μm).
1 13 H NMR and C NMR were obtained in CDCl3 and DMSO-d6 on 400 MHz spectrometers with 13C operating frequencies of 100 MHz and analyzed using MestReNova. Chemical shifts are reported in parts per million (δ) relative to residual chloroform (7.26 ppm for 1H and 77.16 ppm for 13C). Data for 1H NMR spectra are reported as follows: chemical shift (multiplicity, number of hydrogens and assignment). Multiplicity is designated as s (singlet), d (doublet), t (triplet), q
(quartet), p (pentet), or m (multiplet). High-resolution mass spectral (HRMS) data was obtained using electrospray ionization (ESI) and the data was analyzed using MassLynx.
26 2. 2. 1 Experimental Methods
8-Bromo-2'-deoxyadenosine (4)
Bromine (9.6 ml, 194.72 mmol) was added to freshly prepared 1M acetate buffer, pH 5.4 (190 ml) and stirred for 24 hours until completely dissolved. The bromine solution was added drop-wised to a slurry of 2’-deoxyadenosine (10.0 g, 37.13 mmol) in acetate buffer (100 ml). The reaction was monitored by TLC and was completed after 90 min. Solid sodium bisulfite was added to destroy excess bromine, then the reaction mixture was neutralized with 2N NaOH. The reaction mixture was then allowed to cool at -20 °C for 30 minutes. After this time the reaction mixture was filtered and washed with cold water and dried to yield a beige powder 2 (8.5 g, 69 %). TLC
(CH2Cl2/MeOH, 80:20): Rf 0.56.
+ + HRMS (ESI ) m/z calc. for C10H12BrN5O3 [M+H] , 330.1380; found, 330.0188.
1 H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H,), 7.50 (s, 2H), 6.30 (t, J = 8.0, 6.4 Hz, 1H), 5.34
(d, J = 4.2 Hz, 1H), 5.28 (dd, J = 7.7, 4.4 Hz, 1H), 4.49 (dt, J = 6.6, 3.2 Hz, 1H), 3.89 (td, J = 4.7,
2.5 Hz, 1H), 3.66 (dt, J = 11.8, 4.5 Hz, 1H), 3.49 (ddd, J = 12.2, 7.8, 4.9 Hz, 1H), 3.25 (ddd, J =
13.6, 8.0, 6.0 Hz, 1H), 2.20 (ddd, J = 13.1, 6.5, 2.7 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 155.38, 152.63, 150.38, 127.21, 120.17, 88.80, 86.83, 71.62,
62.58, 37.46.
8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (5)
8-bromo-2’-deoxyadenosine, 4 (5.8 g, 17.508 mmol) and imidazole (5.685 g, 83.5175 mmol) were suspended in 15 ml of dry DMF. The reaction mixture was allowed to stir for 10 minutes. After this time, tert-butyldimethylsilyl chloride (7.26 g, 48.15 mmol) was added to the stirring mixture at room temperature. The solution turned transparent within 10-15 minutes and was allowed to stir for an additional 12-16 hours at room temperature. The reaction was monitored by TLC (90/10
27 CH2Cl2/MeOH). The solvent was evaporated under pressure and purified on silica gel column chromatography with a step gradient of methanol (0-6%) in DCM as the mobile phase. The product was isolated as light yellow solid (8.9 g, 90%).
+ + HRMS (ESI ) m/z calc. for C22H40BrN5O3Si2 [M+H] , 559.1833; found 560.0100
1H NMR (400 MHz, Chloroform-d) δ 8.23 (s, 1H), 6.38 (s, 2H), 6.34 (t, 1H), 4.86 (dt, J = 6.1, 3.9
Hz, 1H), 4.06 – 3.77 (m, 2H), 3.77 – 3.45 (m, 2H), 2.22 (ddd, J = 13.1, 7.0, 4.2 Hz, 1H), 0.93 (s,
9H), 0.81 (s, 9H), 0.13 (s, 6H), -0.02 (s, 3H), -0.06 (s, 3H).
8-Bromo-3', 5'-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2'-deoxyadenosine (6)
8-Bromo-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyadenosine 5 (9.8 g, 17.442 mmol) and 4- dimethylaminopyrine (42.2 mg, 0.3488 mmol) was dissolved in 20 ml of anhydrous pyridine. 4,
4'-dimethoxytrityl chloride (8.9 g, 26.24 mmol) was added to the solution at room temperature.
The reaction mixture was then allowed to stir at room temperature for 18 hours. After this time,
TLC analysis indicated complete consumption of 5. The reaction mixture was concentrated in vacuo and the crude reaction mixture was purified on Al2O3 column chromatography with a step gradient of (0-12%) ethyl acetate (EtOAc) in hexanes. The product was isolated as a foamy and white solid (13.83 g, 92%).
+ + HRMS (ESI ) m/z calc. for C43H58BrN5O5Si2 [M+H] , 861.0423; found 862.3206
1 H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.46 – 7.06 (m, 8H), 6.94 – 6.66 (m, 5H), 6.34 (t, J
= 6.7 Hz, 1H), 4.98 – 4.76 (m, 1H), 4.05 – 3.84 (m, 2H), 3.80 (s, 7H), 3.77 – 3.52 (m, 2H), 2.22
(ddd, J = 13.0, 7.0, 4.3 Hz, 1H), 0.95 (s, 9H), 0.83 (s, 9H), 0.15 (s, 6H), -0.00 (s, 3H), -0.06 (s,
13 3H). C NMR (101 MHz, CDCl3) δ 158.53, 158.41, 158.32, 152.99, 152.90, 152.56, 151.96,
151.83, 149.83, 149.52, 145.32, 145.14, 144.90, 142.33, 138.29, 137.37, 136.37, 136.11, 130.26,
130.15, 130.12, 128.83, 128.31, 128.23, 127.88, 127.76, 127.72, 126.82, 126.79, 120.05, 113.20,
28 113.16, 113.05, 110.22, 108.06, 87.70, 86.49, 86.24, 85.01, 72.18, 72.12, 70.66, 62.50, 59.01,
55.21, 36.89, 25.98, 25.90, 25.78, 25.74, 18.36, 18.08, -4.63, -4.68, -5.39, -5.45.
General procedure for Optimization of Buchwald-Hartwig cross coupling reaction.
8-bromo-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyadenosine 5 (0.098 g, 0.1745 mmol) or 8- bromo-3',5'-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2'-deoxyadenosine 6 (0.150 g,
0.1745 mmol ), 6-aminochrysene ( 0.064 mg, 0.263 mmol ), Pd (0) catalyst (4 mol %) and rac-
2,2’-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP, ) ( 12.5 mg, 0.02 mmol) were suspended in
5 ml of anhydrous solvent (see Table 1). The solution was degassed by purging with argon for 1h.
The reaction flask was then heated under argon at 85 – 100 °C for 1h. After this time sodium tert- butoxide, NaOtBu or cesium carbonate, Cs2CO3 (0.263 mmol) was added and the reaction stirred at 85 – 100°C for an additional 1-2 hours. After this time, the reaction was monitored via TLC for consumption of compound 5 or 6. The reaction was then cooled, diluted with CH2Cl2 and filtered through Celite ®. The filtrate was allowed to evaporate in vacuo. For substrate 5, the crude mixture was purified on silica gel column chromatography with a step gradient (0 – 5%) methanol in dichloromethane. For substrate 6, the crude mixture was purified on Al2O3 column chromatography with a step gradient of EtOAc (0-10%) in hexanes as the mobile phase.
8N-(6-aminochrysene)-3',5'-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2'- deoxyadenosine (7b)
8-Bromo-3',5'-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2'-deoxyadenosine 6 (0.595 g,
0.693 mmol ), 6-aminochrysene ( 0.250g, 1.04 mmol ), palladium(II) acetate Pd(OAc)2 (6 mg,
0.0276 mmol) and rac-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP) (50 mg, 0.080 mmol) were suspended in 10 ml anhydrous toluene. The solution was degassed by purging with
29 argon for 1 h. The reaction flask was then heated under argon at 90°C for 1 h. After this time cesium carbonate, Cs2CO3 (0.366 g, 1.04 mmol) was added and the reaction stirred at 100°C for an additional 1hour. The reaction was cooled, diluted with ether and filtered through Celite ®.
The filtrate was allowed to evaporate under reduced pressure. The crude mixture was purified on aluminum oxide column chromatography with a step gradient of EtOAc (0-10%) in hexanes as the mobile phase. The product was isolated as a dark green solid (0.603g, 85%).
+ + HRMS (ESI ) m/z calc. C61H70N6O5Si2 [M+H] , 1023.5025.; found, 1023.5056
1 H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.74 (d, J = 8.3 Hz, 1H),
8.70 (d, J = 9.2 Hz, 1H), 8.61 (s, 1H), 8.16 (dd, J = 8.1, 1.5 Hz, 1H), 8.03 – 7.92 (m, 3H), 7.81 –
7.54 (m, 4H), 7.42 – 7.35 (m, 2H), 7.33 – 7.29 (m, 2H), 7.28 – 7.17 (m, 5H), 6.85 – 6.70 (m, 4H),
6.65 (s, 1H), 6.53 (t, J = 7.8, 5.4 Hz, 1H), 4.23 (q, J = 3.3 Hz, 1H), 3.99 (dd, J = 11.4, 3.6 Hz, 1H),
3.91 – 3.80 (m, 1H), 3.77 (s, 6H), 3.21 (ddd, J = 13.4, 7.8, 5.9 Hz, 1H), 2.51 (ddd, J = 13.0, 5.5,
2.9 Hz, 1H), 0.96 (s, 8H), 0.61 (s, 8H), 0.16 (d, J = 4.6 Hz, 6H), -0.18 (s, 3H), -0.30 (s, 3H).13C
NMR (101 MHz, Chloroform-d) δ 158.07 , 151.59 , 149.54 , 149.20 , 145.89 , 138.18 , 133.40,
132.49 , 131.49 , 130.09 , 128.87 , 128.83 , 128.43 , 127.73 , 126.82 , 126.70 , 126.54 , 126.51 ,
126.30 , 126.11 , 125.33 , 123.99 , 123.53 , 121.38 , 121.01 , 118.71 , 113.20 , 113.04 , 88.35 ,
85.60 , 72.43 , 70.31 , 63.02 , 55.16 , 39.79 , 25.84 , 25.65 , 18.24 , 18.07 , -4.51 , -4.72 , -5.71 , -
5.76 .
8N-(6-aminochrysene)-3',5'-O -bis(tert-butyldimethylsilyl)-2'-deoxyadenosine (7a)
Method 1
Fully protected coupled substrate, 8N-(6-aminochrysene)-3',5'-O-bis(tert-butyldimethylsilyl)-N6- dimethoxytrityl-2'-deoxyadenosine, 7b (2.0 g, 1.95 mmol) was dissolved in 5.0 mL dichloromethane and to this added 1M zinc bromide solution in methanol: dichloromethane (1:1)
30 (6 mL). The reaction was stirred for 0.5 h and was monitored for completion by TLC (90/10
DCM/MeOH, v/v). The reaction was quenched with aqueous sodium bicarbonate and extracted with dichloromethane. The organic layer was combined and dried with sodium sulfate. The solvent was evaporated under reduced pressure and crude product was purified by silica gel flash column chromatography with a step gradient of 0-10% methanol in dichloromethane to afford compound
6 as a beige powder (1.05 g, 75%).
Method 2
8-Bromo-3', 5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyadenosine 5 (0.098 g, 0.1745 mmol), 6- aminochrysene ( 0.064 mg, 0.263 mmol ), tris(dibenzyldeneacetone)dipalladium (Pd2(dba)3 (6.4 mg, 0.007 mmol) and rac-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP) ( 12.5 mg, 0.02 mmol) were suspended in 8 ml diethoxy ethane (DME). The solution was degassed by purging with argon for 1 h. The reaction flask was then heated under argon at 85 °C for 1 h. After this time cesium carbonate, Cs2CO3 (86 mg, 0.263 mmol) was added and the reaction stirred at 85°C for an additional hour. The reaction was cooled, diluted with dichloromethane and filtered through
Celite®. The filtrate was allowed to evaporate under reduced pressure. The crude mixture was purified on silica gel column chromatography with a step gradient (0 – 5%) methanol in dichloromethane. The product 7a was isolated as a beige powder (71 mg, 56%)
+ + HRMS (ESI ) m/z calc. C40H52N6O3Si2 [M+H] , 721.37; found, 721.3729
1 H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H), 8.86 (d, J = 8.4 Hz, 1H), 8.74 (dd, J = 23.0, 8.6 Hz,
3H), 8.42 (s, 1H), 8.25 (d, J = 1.9 Hz, 1H), 8.16 (d, J = 7.9 Hz, 1H), 8.00 (t, J = 9.4 Hz, 3H), 7.81
– 7.60 (m, 5H), 6.64 (dd, J = 8.4, 5.4 Hz, 1H), 5.30 (d, J = 15.9 Hz, 1H), 4.67 (dd, J = 5.9, 2.9 Hz,
1H), 4.22 (d, J = 2.9 Hz, 1H), 4.05 (dd, J = 11.5, 3.0 Hz, 1H), 3.88 (dd, J = 11.5, 2.8 Hz, 1H), 3.12
(dt, J = 14.0, 7.1 Hz, 1H), 2.54 – 2.40 (m, 1H), 0.98 (s, 9H), 0.56 (s, 9H), 0.17 (d, J = 8.7 Hz, 6H),
31 13 -0.24 (s, 3H), -0.33 (s, 3H). C NMR (101 MHz, CDCl3) δ 158.53 , 158.32 , 152.99 , 151.96 ,
145.32 , 142.33 , 137.37, 136.11 , 130.15 , 130.12 , 128.83 , 128.23 , 127.88 , 127.76 , 126.82 ,
126.79 , 120.05 , 113.20 , 113.16 , 113.05 , 110.22 , 108.06 , 87.70 , 85.01 , 72.12 , 70.66 , 62.50
, 59.01 , 55.21 , 36.89 , 25.90 , 25.78 , 25.74 , 18.36 , 18.08 , -4.63 , -4.68 , -5.39 , -5.45.
8N-(aminochrysene)-2'-deoxyadenosine (3)
8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-2'-deoxyadenosine 7a (200 mg,
0.2772 mmol) was dissolved in 3 ml of anhydrous tetrahydrofuran. 0.416 ml of 1M tetrabutylammonium fluoride in tetrahydrofuran was added and the reaction was allowed to stir for 24 hours under N2. After this time, the solvent was evaporated under reduced pressure and the crude product was purified via silica gel column chromatography with methanol (0-5%) in dichloromethane. The product was isolated as a light brown solid (116 mg, 85%).
+ + MS (ESI ) m/z calc. C28H24N6O3 [M+H] , 493.53; found, 493.53
1 H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 9.01 (d, J = 8.4 Hz, 1H), 8.89 (d, J = 7.4 Hz, 2H),
8.84 (d, J = 8.2 Hz, 3H), 8.19 (d, J = 8.2 Hz, 1H), 8.10 (t, J = 6.8 Hz, 2H), 8.02 (s, 1H), 7.84 –
7.66 (m, 4H), 4.14 – 3.99 (m, 1H), 3.81 (d, J = 11.9 Hz, 1H), 3.10 (td, J = 15.5, 14.4, 8.3 Hz, 1H),
13 2.30 (dt, J = 12.9, 6.9 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 153.75, 150.12, 150.02, 149.66,
135.98, 132.38, 131.56, 130.00, 128.90, 128.53, 127.64, 127.43, 127.24, 127.12, 126.32, 124.26,
124.18, 124.04, 121.74, 117.26, 116.87, 88.27, 84.32, 71.90, 62.20, 58.00, 55.38, 38.79.
N6-Benzoyl-8N-(aminochrysene)-2'-deoxyadenosine (8)
8N-(6-aminochrysene)-3',5'-O -bis(tert-butyldimethylsilyl)-2'-deoxyadenosine, 7a
(445mg, 0.617 mmol) was dissolved in anhydrous pyridine (5 mL) under an atmosphere of nitrogen and benzoyl chloride (0.108 mL, 0.9255) was added dropwise. The reaction mixture was stirred for 4 h at room temperature. It was then diluted with dichloromethane (10 mL) and washed
32 with saturated sodium hydrogen carbonate solution, and the aqueous layer was extracted twice with dichloromethane. After removal of the dichloromethane under reduced pressure, morpholine
(0.134 mL, 1.5425 mmol) was added and the resulting mixture was stirred at room temperature for
2 h. After this time, the reaction mixture was diluted with dichloromethane (10mL) and then washed twice with 0.5M sodium dihydrogenphosphate solution. The aqueous layer was extracted three times with dichloromethane. After complete removal of the dichloromethane under reduced pressure, 100 mg, 0.1386 mmol of crude product was dissolved in 3 ml of anhydrous tetrahydrofuran. 0.416 ml of 1M tetrabutylammonium fluoride in tetrahydrofuran was added and the reaction was allowed to stir for 24 hours under N2. After this time, the solvent was evaporated under reduced pressure and the crude product was purified via silica gel column chromatography with methanol (0-10%) in dichloromethane. The product was isolated as a yellow solid (70 mg,
85%).
HRMS (ESI+) m/z calc. C35H28N6O4 [M+H] +, 597.2250; found, 597.2234 and [M+Na]+
1 H NMR (300 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.99 (d, J = 7.8 Hz,
1H), 8.83 (d, J = 8.9 Hz, 2H), 8.58 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 6.6 Hz, 5H), 7.79
(s, 0H), 7.59 (dt, J = 14.5, 7.4 Hz, 1H), 7.43 (dt, J = 14.4, 7.6 Hz, 3H), 6.82 (t, J = 7.1 Hz, 1H),
5.48 (d, J = 4.2 Hz, 2H), 4.57 (s, 1H), 4.12 (s, 1H), 3.91 – 3.59 (m, 3H), 3.27 – 2.97 (m, 1H), 2.42
13 (dd, J = 12.9, 6.0 Hz, 1H). C NMR (75 MHz, DMSO-d6) δ 151.51 , 148.95 , 145.54 , 134.07 ,
134.00, 132.54 , 132.42 , 131.26 , 130.18 , 128.84 , 128.75 , 128.36 , 127.60 , 127.35 , 127.20 ,
127.05 , 126.96 , 125.69 , 124.33 , 124.06 , 123.33 , 121.64 , 115.19 , 88.45 , 84.85 , 71.77 , 62.02
33 N6-Benzoyl-8N-(6-aminochrysene)-5'-O-dimethoxytrityl-2'-deoxyadenosine (9)
The N6-benzoylated C8-arylamine-dA adduct 8 (0.295g, 0.494 mmol) was dissolved in anhydrous pyridine (6 mL) under a nitrogen atmosphere and 4,4’-dimethoxytrityl chloride (0.177g, 0.522 mmol) and silver nitrate (0.089g, 0.522 mmol) were added. The mixture was stirred at room temperature until the reaction was complete (18 h). The reaction mixture then diluted with dichloromethane (10 mL) and washed with saturated sodium hydrogencarbonate solution and brine. The aqueous layer was extracted twice with dichloromethane. The organic layers were combined, dried over sodium sulfate, and filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 0-3% methanol in dichloromethane to afford the desired DMT protected 7 as a yellow foam, (328 mg,
74%).
+ + HRMS (ESI ) m/z calc. C56H46N6O6 [M+H] , 899.3552; found, 899.3532
1 H NMR (500 MHz, CDCl3) δ 9.31 (s, 1H), 8.76 (dd, J = 7.5, 1.3 Hz, 1H), 8.72 – 8.73 (m, 1H),
8.44 (s, 1H), 8.23 (s, 1H), 8.16 (dd, J = 7.8, 1.3 Hz, 1H), 8.05 – 8.03 (m, 2H), 8.00 – 7.94 (m, 1H),
7.89 (d, J = 9.2 Hz, 1H), 7.72 – 7.66 (m, 2H), 7.66 – 7.62 (m, 2H), 7.54 – 7.42 (m, 3H), 7.42 –
7.38 (m, 2H), 7.37 (d, J = 1.4 Hz, 1H), 7.33 – 7.27 (m, 2H), 7.27 – 7.20 (m, 1H), 7.13 – 7.07 (m,
4H), 6.87 – 6.81 (m, 4H), 6.60 (ddd, J = 4.0, 2.4, 0.9 Hz, 1H), 6.30 (s, 1H), 4.23 – 4.18 (m, 2H),
3.82 (s, 6H), 3.68 (qd, J = 12.0, 3.1 Hz, 2H), 2.56 (dt, J = 12.5, 3.3 Hz, 1H), 2.31 (dt, J = 12.3, 5.1
13 Hz, 1H), 1.91 (s, 1H). C NMR (126 MHz, CDCl3) δ 165.57, 165.06, 158.21, 153.17, 152.09,
151.83, 149.38, 144.96, 144.53, 143.84, 136.17, 135.10, 135.01, 134.11, 132.71, 132.45, 132.10,
131.22, 130.09, 129.83, 129.77, 129.33, 128.49, 128.38, 128.24, 128.15, 127.68, 127.64, 127.01,
126.80, 126.73, 126.41, 125.91, 125.50, 124.80, 123.68, 123.51, 123.13, 122.01, 121.90, 121.10,
34 120.62, 115.11, 112.85, 110.01, 86.81, 86.48, 85.69, 71.79, 67.62, 63.37, 63.30, 54.93, 53.49,
53.07, 53.04, 53.02, 53.00, 52.93, 52.85, 52.83, 52.81, 52.79, 52.77, 40.96, 39.45, 0.01.
N6-Benzoyl-8N-(6-aminochrysene)-5'-O-dimethoxytrityl-2'-deoxyadenosine-3'-O-
(cyanoethyl-N,N'-diisopropyl-phosphoramidite (10)
This reaction was performed in a glove bag. N6-Benzoyl-8N-(6-aminochrysene)-5'-O- dimethoxytrityl-2'-deoxyadenosine 9 (0.400 g, 0.444 mmol), N, N-diisopropylethylamine (0.155 mL, 0.888 mmol) was dissolved in anhydrous dichloromethane (6 mL) under argon. 2-cyanoethyl-
N, N-diisopropylchlorophosphoramidite (0.105 mL, 0.444 mmol) was added and the reaction mixture stirred for 15 minutes. After this time, additional N, N-diisopropylethylamine (0.155 mL,
0.888 mmol) was added to the solution and the reaction stirred for an additional 0.5 h. After this time, the reaction mixture was diluted with dichloromethane (20 mL) and saturated sodium bicarbonate (25 mL). The organic layer was retained, and the aqueous layer was extracted twice with dichloromethane (2 x 15 mL). The organic layers were combined, washed with brine and dried over sodium sulfate. The solvent was concentrated under reduced pressure (2 mL). The solution was added dropwise to a stirring solution of 220 mL hexanes. The resulting precipitate was collected and further purified on basified silica gel flash chromatography with a step gradient
(0 -5%) methanol in dichloromethane containing 1% triethylamine. The product was isolated as a yellow solid (0.297 g, 61%)
HRMS (ESI+) m/z calc. C65H63N8O7P [M+H] +, 1099.4636; found, 1099.4607
31 P NMR (162 MHz, CD2Cl2) δ 149.06, 149.01, 148.57, 148.51.
35 2. 2. 2 Synthesis of Site-Specifically Modified Oligodeoxynucleotides Phosphoramidite 10 was used to synthesize modified oligodeoxynucleotides as per manufacture’s instruction. However, the time of the coupling step was repeated extended to 15-min for the incorporation of modified phosphoramidite with total coupling efficiencies of > 95%. The unmodified and complementary oligodeoxynucleotide sequences were purchased from Integrated
DNA Technologies, Inc in Coralville, Iowa, USA.
2. 2. 3 Purification of Site-Specifically Modified Oligodeoxynucleotides The modified oligonucleotides were purified by reverse-phase HPLC and denaturing polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified modified and unmodified (control) oligodeoxynucleotides were confirmed via phosphorylation with T4 polynucleotide kinase in the presence of [g32P] adenosine triphosphate and subsequent PAGE analysis. The modified oligodeoxynucleotides were further characterized by ESI mass spectrometry. HRMS data are given in Table 3.
HPLC Purification
The crude oligodeoxynucleotides were purified using reversed phase HPLC utilizing a C18 – semi prep column (Phenomenex Gemini –NX 5μ C18 (250 × 10 mm)) with an eluent of 0.1 M NH4OAc in ACN-H2O (solvent A) and 80% ACN-20% H2O (solvent B) with a linear gradient (0 to 50 min,
2% to 30%) at a flow rate of 3.0 mL/ min monitored at both 254 nm and 340 nm. The purified oligomers were collected in several 1.5 mL microcentrifuge tubes and filtered through Amicon ultracel 3K filters. The filtrates were collected and dried in a SpeedVac.
PAGE Purification
After HPLC purification, the oligomers were purified by denaturing polyacrylamide gel electrophoresis. Modified and unmodified oligomers were loaded onto 20% PAGE (50 cm length)
36 containing 8M urea. The gel was run at 2250 V (45V/cm2) for 5~6 hours at room temperature until the leading dye bromophenol blue (which runs approximately with a 6mer) reached the bottom of the gel. The desired oligomer-containing band was visualized using a UV (254 nm) light and excised from the gel. The oligonucleotides were extracted from the excised gel by Spin-X (Costar
Corning) columns and eluted with water, and subsequently desalted by passing through Sep-Pak
C18 cartridges (Waters) with acetonitrile:water (60:40) . The elute from each sample was dried in a speed-vac and the concentration was determined by UV absorbance measurements. The homogeneity of the purified modified and unmodified (control) oligodeoxynucleotides were confirmed via phosphorylation with T4 polynucleotide kinase in the presence of [g32P] adenosine triphosphate and subsequent PAGE analysis.
37 2. 3 Results and Discussion
In order to synthesize the C8-dA adduct of 6-NC in any desired DNA sequence, we employed a total synthesis approach. This approach involves the synthesis of the modified 3’phosphoramidite monomer which can be site-specifically incorporated in any desire oligodeoxynucleotide sequence via solid-phase DNA synthesis. While many C8-dG adducts by different polycyclic aromatic
47,61–63 amines and NO2-PAHs have been synthesized and incorporated in DNA by this method, there are only a few reports on the synthesis of dA adducts. Notably, synthesis of several polycyclic aromatic hydrocarbons (PAH) derived DNA adducts at the 6 position of dA have been accomplished,62,64,65 but only Meier46 and Takamura17 have incorporated C8-arylamine-modified dA adducts in DNA by total synthesis.46 They used the Pd-catalyzed Buchwald-Hartwig C-N bond forming strategy to incorporate phenylamine derivatives and 4-aminobiphenyl at the C8 position of dA.
R NH2 NH2 HN N N N N N Br N 1. TBDMSCl, imidazole Br N N N Br2, NaOAc Buffer N pyridine, 16 h @ RT N HO N HO (pH 5.4) TBDMS-O O O O 2 h @ RT 2. DMTrCl, 4-DMAP pyridine, 18 h @RT OH OH O-TBDMS 2’-dA 4, (81%) R = H 5, (95%) R = DMTr 6, (92%) Scheme 3.Preparation of Fully Protected Br-dA.
Selective bromination at C8 position of dA was achieved using Br2 in acetate buffer (pH 5.4.)
Protection of the 3’, 5’ hydroxyls and exocyclic amine is necessary to avoid unwanted reaction in the Buchwald-Hartwig cross coupling reaction. Thus, silylation of 3’, 5’ hydroxyls moieties were achieved using tert-butyldimethylsilyl chloride. The N6-exocyclic amine was protected using 4,4′- dimethoxytrityl chloride (DMTCl) in pyridine with catalytic amounts of 4-dimethylaminopyridine
(DMAP), which provided fully protected dA substrate 6 for coupling.
38 2. 3. 1 Buchwald-Hartwig Cross-Coupling Reaction Optimization We envisioned performing Buchwald-Hartwig cross-coupling reaction without protection of the N6 exocyclic amine as previously reported46,49. In order to increase the efficiency of the key palladium catalyzed Buchwald-Hartwig cross coupling reaction; a series of reaction conditions with respect to N6 protected or unprotected substrate, catalyst, base, solvent and temperature were screened to determine the optimal reaction conditions (Table 2). Previous work have demonstrated that Pd2(dba)3/NaOtBu were good catalyst/base combo; while combination of Pd(OAc)2/Cs2CO3 work best together46,50. Thus, Buchwald-Hartwig optimization were done with this framework in mind. Initially N6 exocyclic free amine substrate, 5 reacted with 6-aminochrysene (6-AC) with combinations of Pd2(dba)3/NaOtBu in toluene as solvent at 100 ˚C. The desired product was isolated with 45% yield (Table 2, entry 1). Identical reaction conditions except with combination of Pd(OAc)2/Cs2CO3 furnished the desired product with increased yield of 56% (Table 1, entry 2).
Each reaction shown here was carried out for 3-4 h. Longer reaction times (up to 24 h) led to significant tailing on TLC for reactions carried out at 100 °C in toluene, which is usually indicative of degradation of the product. In 1,2-DME (performed at 85°C) this tailing was not observed, and the reactions were allowed to run overnight. However, longer reactions times did not result in improvement in reaction yields.
For the Pd-catalyzed coupling of protected dG with polyaromatic amines, Gillet and Schärer showed that N2-DMT-protected dG provides excellent yield of the adduct,47 which we have used successfully for the synthesis of dG-1-aminopyrene adduct.50 So, to improve the coupling efficiency, the N6 exocyclic amine was protected using 4,4′-dimethoxytrityl chloride (DMT-Cl) in pyridine with catalytic amounts of 4-dimethylaminopyridine (DMAP) and then the N6 DMT protected Br-dA nucleoside, 6 was subjected to the Pd-catalyzed coupling. Gratifyingly, N6 exocyclic amine DMTr protected substrate, 6 coupled with 6-AC with combination of
39 o Pd(OAc)2/Cs2CO3 in toluene to furnish the desired product with 85% yield at 100 C for 3 h (Table
2). For the DMT protected Br-dA nucleoside (6) also longer reaction time did not improve the yield. We believe that the improved yields with N6 DMT protected substrate in the Buchwald-
Hartwig cross coupling reactions can be attributed to the increased solubility of the substrate in toluene (and DME) facilitated by the non-polar DMT group.
R HN R HN N N Br N N Pd (0), Base HN N N + TBDMS-O Solvent, T (ºC) N N O TBDMSO O NH2 O-TBDMS O-TBDMS
5 R = H (7a) R = DMTr (7b) Entry R Catalyst Base Ligand Solvent Temp. %Yield (4 mol %) (1.5 equiv) (12 mol%) (˚C) 1 H Pd2(dba)3 NaOtBu BINAP Toluene 100 45 2 H Pd(OAc)2 Cs2CO3 BINAP Toluene 100 56 3 H Pd2(dba)3 NaOtBu BINAP DME 85 52 4 H Pd(OAc)2 Cs2CO3 BINAP DME 85 42 5 DMT Pd2(dba)3 NaOtBu BINAP Toluene 100 75 6 DMT Pd(OAc)2 Cs2CO3 BINAP Toluene 100 85 7 DMT Pd2(dba)3 NaOtBu BINAP DME 85 68 8 DMT Pd(OAc)2 Cs2CO3 BINAP DME 85 61 aAll reactions were carried out for 3-4 h. Table 2. Optimization of Buchwald-Hartwig cross coupling reaction.
40
P P HN HN Pd(BINAP) 2 N N N N Br HN Ar Br = Ar NR1R2 N N N N P O O P O O (BINAP)Pd [0] O O P P Reductive Elimination Oxidative Addition
Catalytic Br 2+ BINAP ᵦ-hydride elimination 2+ Pd R2R1N BINAP Cycle (if possible) Pd Ar Ar
O + CsBr + Cs R2 O OH Base Mediated N R1 Palladium-Amide H Bond Formation R H Ar 2+ BINAP 2 N = Pd R1 NH Br 2
O Cs+ Cs+ HO O
Figure 11. Mechanism of Buchwald-Hartwig cross coupling reaction of protected 8-bromo-2’- deoxyadenosine and 6-aminochrysene (6-AC)
2. 3. 2 Synthesis of Modified Phosphoramidite Monomer With the optimized conditions in hand and an efficient strategy to access coupled product 7b;
6 N -excocyclic amine was deprotected using a 1 M solution of ZnBr2 to furnish 7a. To enable solid phase DNA synthesis, N6 amine was then protected with base-labile benzoyl group using benzoyl chloride in pyridine followed by treatment with morpholine to afford N6-benzoylated product; desilylation of the 5’- and 3’-hydroxyl was achieved using tetrabutylammonium fluoride (TBAF) in THF to furnish 8. To initiate DNA synthesis the 5’postion was protected with acid-labile DMTr,
9. The corresponding 3’-phosphoramidite was prepared using 2-Cyanoethyl-N,N- diisopropylchlorophosphoramidite to give phosphoramidite monomer 10 (Scheme 6).
41
DMTr HN NH2 NH2 N N N N N HN a HN N b HN N N N N TBDMSO TBDMSO N N O O RO O
OTBDMS OTBDMS OR R = H; N-(dA-8-yl)-6-AC (3) 7b 7a R = TBDMS (7a)
c, d
O
HN Ph O O N N HN HN Ph HN Ph N N DMTrO N N N N O f HN e HN N N N N CN DMTrO HO O P O O N OH OH 10 9 8 ( from 7a) Scheme 4. Synthesis of Corresponding 3’-Phosphoramidite 10 a) 1M ZnBr2 in 1:1 solution.
MeOH:CH2Cl2, 30 mins, 75%; b) 1M TBAF in THF, RT, 24h, 85%; c) Benzoyl chloride (BzCl), pyridine, RT followed by treatment with morpholine, 2h d) 1M TBAF in THF, RT, 24h, 85%; e)
4,4-dimethoxytritylchloride (DMTCl), pyridine, AgNO3, RT 24h, 74% f) 2-Cyanoethyl-N,N- diisopropylchlorophosphoramidite, DIEA, CH2Cl2, RT, 1h, 61%.
2. 3. 3 Synthesis of Oligodeoxynucleotides Containing N-(dA-8-yl)-6-AC Adduct Fully protected N-(dA-8-yl)-6-AC phosphoramidite (10) was incorporated in the 12 and 15- mer oligodeoxynucleotides with total coupling efficiency of > 95%. Oligodeoxynucleotide deprotection was done at 55°C with ammonium hydroxide for 24h to furnished modified oligodeoxynucleotides containing N-(dA-8-yl)-6-AC adduct (Scheme 7). During deprotection,
0.25M 2-mercaptoethanol was added to avoid oxidative degradation. These modified oligodeoxynucleotides were purified by reverse-phase HPLC and PAGE and characterized by high resolution electrospray ionization mass spectrometry in negative mode (Table 3).
42 CT Adduct CT Adduct
mer mer mer mer 12 12 15 15 (-) Deprotection Cleavage Desalting
Purification • HPLC • PAGE LC-MS analysis (+) CPG CPG CPG CPG 1 2 3 4
Scheme 5. Modified oligodeoxynucleotides deprotection and purification
Oligodeoxynucleotides Calculated (M-3H)3- Observed (M-3H) 3- 5'-GTG CAT GTT TGT-3' 3679.622 3679.627 5'-GTG CA*T GTT TGT-3' 3921.922 3921.7274 5'-GCCCTCAACAAGATG-3' 4542.800 4542.8010 5'-GCCCTCAA*CAAGATG-3' 4784.100 4784.9298
Table 3. HRMS-ES mass for modified and unmodified (control) oligodeoxynucleotides
The 12-mer sequence was taken from part of p53 tumor suppressor gene, 5’-GTG CG*T
GTT TGT-3’ where the underlined part is codon 273, which is frequently mutated in many different types of human cancers. We replaced G* with A* so that mutations from A* can be compared with the G adduct. The 15-mer sequence (Table 3), 5'-GCC CTC AA*C AAG ATG-3' was taken from another part of p53, which include codon 131 (underlined). Codon 131 is one of the rare mutation hotspots in which an adenine is mutated. This was discovered from mutation spectrum by aristolochic acid, a naturally occurring nitroaromatic compound and a dietary contaminant.22,24,62
43 O NH O O O O O O O O O O O O O O O O HN Ph 5’ O P G P C P C P C P T P C P A P A P C P A P A P G P A P T P G P O 3’ O O O O O O O O O O O O O O O O N N HN 15 mer oligonucletide N N DMTrO DNA O Synthesis NH O O O O O O O O O O O O O O CN P 5’ O P G P T P G P C P A P T P G P T P T P T P G P T P O 3’ N O O O O O O O O O O O O O 12 mer oligonucleotide C8-dA-6AC Phosphoramidite Monomer (10) Figure 12. Structures of modified oligodeoxynucleotides.
2. 4 Conclusion
In summary, we have reported an efficient strategy for the synthesis of C8-6-nitrochrysene adduct of 2‘-deoxyadenosine. The 6-aminochrysene carcinogen was introduced at the C8 site of 2’-dA using Buchwald−Hartwig palladium catalyzed cross-coupling chemistry. This optimized strategy provides efficient and convenient access to N-(dA-8-yl)-6-AC adduct. The corresponding
3’phosphoramidite adduct of N-(dA-8-yl)-6-AC were site-specifically incorporated into oligodeoxynucleotides via. solid-phase DNA synthesis.
44
Chapter 3
Conformational Preference and Stability of 6-Nitrochrysene C8-2’Deoxyadeosine Adduct
†Powell, B.V.; Basu, A.K. “6-Nitrochrysene-Derived C8-2’-Deoxyadenosine Adduct: Synthesis of Site-Specific Oligodeoxynucleotides and Mutagenicity in Escherichia coli. 2020 Chem Res Toxicol [In Press]
45
3. 0 Abstract
O
NH2 HN Ph A* N N N N HN HN N N N N HO DNA 2'dA O DMTr O O A* Synthesis
OH O O P CN ROESY NMR Conformational N Analysis Conformation Analysis
Carcinogen-DNA adducts that derive from covalent modifications of DNA nucleobase by nitro polycyclic aromatic compounds are known to form bulky DNA adducts. These adducts can assume various conformations in DNA helix which can be responsible for inducing a variety of mutagenic outcomes. We investigated the conformational preference of the C8-
2’deoxyadenosine nucleoside that derives from 6-nitrochrysene modification, N-(dA-8-yl)-6-AC.
The adducted nucleoside showed syn conformational preference by NMR spectroscopy and DFT calculations. We also investigated the stability of N-(dA-8-yl)-6-AC adducted nucleoside using
UV/Vis spectroscopy and HPLC. We determined that N-(dA-8-yl)-6-AC adduct is stable under in vitro biologically relevant conditions. We further investigated the helical structure of N-(dA-8-yl)-
6-AC oligodeoxynucleotide containing duplexes using circular dichroism spectroscopy. The CD curves of the N-(dA-8-yl)-6-AC containing 12 and 15mer duplexes showed only slight deviation from the B type DNA helical structure of the unmodified duplex.
46 3. 1 Introduction
Nitrogen-linked arylamine C8-dG lesions can adopt three distinct conformations (Figure 13) depending on the orientation about the glycosidic (sugar-base) bond and the location of the bulky aryl ring system within the duplex.66 These adducts are known to possess conformational heterogeneity. This refers to the ability of nucleotides to adopt more than one conformational posture about the glycosidic bond.67–69 The two orientation nucleotides can typically adopt are anti and syn of the nucleobase to the sugar.66 The ability of nucleotides to assume different conformations is believed to be an important factor in mutation outcomes. Carcinogen-DNA adduct possesses altered biophysical properties compared to the precursor nucleoside.70–72 It is believed that the correlation between aryl ring size, adduct planarity, and sequence context regulate distribution in conformational preference. Wetmore and coworkers purports that the distribution in conformational preference of adduct greatly impacts repair propensity and mutational outcomes.66
B-type S-type W-type
major groove major groove O major groove O H2N NH NH2 NH O Aryl NH 2 N N N Aryl NH2 NH N N NH N N 2 N HN O N N N N N N H HN N syn Rd O Rd N dR dR Rd O H2N Rd syn anti minor groove minor groove Aryl minor groove
Figure 13. Depictions of three major conformations induced in N linked C8-aryl-dG adducts
The nucleotide excision repair (NER) efficiency of N-(dA-8-yl)-6-AC adduct (Figure 14) (3) is eight times less that the N-(dG-8-yl)-6-AC (1) in identical sequence context.14 This suggests that
N-(dA-8-yl)-6-AC adduct (3) is potentially more mutagenic than N-(dG-8-yl)-6-AC (1).
47 NH O O O 2 N N N N N NH NH NH N N N H N N H N H N N N N N N NH 2 HO HO H HO HO O O O O NH2
OH OH OH OH
N-(dG-8-yl)-6-AC (1) N-(dA-8-yl)-6-AC (3) 5-(dG-N2-yl)-6-AC (2) N-(dI-8-yl)-6-AC (3’)
Figure 14. Major DNA adducts derived from 6-NC via nitroreduction pathway
In human cells, bulky DNA lesions derived from nucleobase modifications are subjected to NER that attempt to restores the integrity of DNA prior to DNA replication through a ‘cut and patch’ mechanism.4,31 NER in humans involve recognition of helix distorting DNA damages that recruits
NER enzymes.73,74 This process involves as many as 30 distinct proteins in a large complex known as nucleotide excision repairosome. It is believed that local thermodynamic destabilization plays an important role in determining lesion recognition and subsequent excision efficiency.30
Therefore, non-distorting conformers of adducts may escape NER repair, while distorting conformers may be more efficiently repaired. Thus, the equilibrium between conformations might help determine their potential carcinogenicity.68 We hypothesized that N-(dA-8-yl)-6-AC (3) adduct is less distorting that N-(dG-8-yl)-6-AC (1) in an identical sequence context and therefore results in less destabilization and so is poorly recognized by NER enzymes.
To test this hypothesis, we studied the intrinsic conformation of the adducted free nucleoside to establish the preferred conformation of the adducted nucleoside. Interestingly, Sproviero et. al. demonstrated that bulky C8-arylamine-dG prefers syn conformation as adducted nucleosides but prefers anti conformation in duplex DNA. Furthermore, we studied the conformation of N-(dA-8- yl)-6-AC (3) using circular dichroism spectroscopy and molecular dynamics. Furthermore it has been suggested that N-(dA-8-yl)-6-AC adduct (3) undergoes spontaneous deamination to produce
48 a deoxyinosine derivative N-(dI-8-yl)-6-AC.14 We were interested in understanding this facile deamination. To this end, we’ve studied the stability of N-(dA-8-yl)-6-AC under physiological temperature and pH.
49 3. 2 Materials and Methods
3. 2. 1 Synthesis of N-(dA-8-yl)-6-AC nucleoside See 2.2.1 [ Synthesis of compound 3]
3. 2. 2 Synthesis of site-specifically modified oligodeoxynucleotides See 2. 2. 4 3. 2. 3 DFT Calculations
11 NH2 R 7 5 6 θ N φ N 1 N 8 10 H N 4 N 2 R = 5’ 9 3 HO O χ 4’ 1’ 3’ OH 2’
Figure 15. Structures and chemical numbering of N-linked DNA adduct at the C8-site of 2′- deoxyadenosine
To analyze the intrinsic conformational preferences of the adducts, the potential energy surface was mapped as a function of the θ (∠(N9−C8−N10−C11)) and φ (∠(C8−N10−C11−C12)) dihedral angles using nucleobase models and as a function of χ (∠(O4′−C1′−N9−C4)) and θ using nucleoside N-(dA-8-yl)-6-AC adducts.75 Specifically, surfaces were generated using B3LYP/6-
31G(d) optimizations by altering and constraining each dihedral angle from 0° to 350° in 10° increments. B3LYP/6-31G(d) has been successfully used to study the conformational landscape of several DNA adducts.66,68 However, the extended π-system in the adducts investigated in the present work suggest that more accurate modeling of dispersion forces may be required.68
Therefore, all minima identified from the scans were subsequently fully optimized with both
B3LYP and B3LYP-D3(BJ) using the 6-31G(d) basis set for comparison. The similarities in the key structural parameters obtained with both functionals support our use of B3LYP for the
50 computationally intensive scans. Regardless, more accurate relative energies were obtained for the fully B3LYP-D3 optimized minima with B3LYP-D3(BJ)/6-311+ G(2df,p), which include scaled
(0.9813) zero-point vibrational energy (ZPVE) corrections.76
The initial nucleobase models were built using GaussView 5.0 by appending the bulky groups to the template guanine. The nucleoside models were built from the lowest energy con- formation obtained for the nucleobase models by replacing H9 with 2′-deoxyribose in the B-DNA conformation obtained in a previous conformational analysis (i.e., C2′-endo pucker, β
(∠(H−O5′−C5′−C4′)) dihedral angle fixed to 180° and the dihedral angle at the 3′ terminus
(∠(H−O3′−C3′−H3′)) locked to 60°). All DFT calculations were performed using Gaussian 09
(revision D.01).
3. 2. 4 Circular Dichroism Studies The concentration of oligodeoxynucleotides were determined using NanoDrop 2000 spectrophotometer. Equal amount of two complementary strands (5 nmol) were dissolved in buffer [1 mL, NaH2PO4 buffer (10mM), NaCl (140 mM), EDTA (1mM), pH 6.6] and heated to
70°C and then slowly allowed to cool to room temperature to enable annealing of the two strands.
CD measurements were carried out at 25 °C and samples were scanned from 400 to 200 nm at 0.5 nm intervals averaged over 5 s on a ChirascanTM V100.
3. 2. 5 UV/Vis Spectroscopy Stability Studies In 50 mm phosphate-buffered saline (PBS), pH 7.3 (containing DMSO, which was necessary to enhance the solubility). While incubating both compound 3 and 2’deoxyadenosine as control at concentrations of 6mM at 37 °C, aliquots were taken at various time points, diluted with water to a final concentration of 0.3 mM and analyzed by their UV/Vis absorption spectra.77
51 3. 3 Results and Discussion
chrysene-NH C2-Adenine-H Sugar-H1’
7
Figure 16. Proton NMR spectrum of N-(dA-8-yl)-6-AC adducted nucleoside (3) highlighting conformationally relevant peaks
NMR data on C8-arylamine-dA adducts showed that with a single phenyl ring, the preferred conformation of the N-glyosidic bond is anti, but addition of an N-acetyl group replacing the amino hydrogen rotates it to syn conformation.46 To determine syn or anti preference of N-(dA-8-yl)-6-
AC, its conformation was investigated using ROESY-NMR spectroscopy. Figure 16 shows 1H
NMR spectrum with of N-(dA-8-yl)-6-AC adducted nucleoside with conformationally relevant peak assignment, while Figure 17 shows the ROESY NMR spectrum with a cross-peak between
H1' proton of the sugar and 6-AC amine tethered to the C8 site of dA, indicating a preference for syn conformation. Furthermore, no cross-peaks were observed between the H2 proton on nucleobase and the H1' proton on the sugar, typically observed for anti-conformation. These results indicate a preference of N-(dA-8-yl)-6-AC to exist in the syn conformation of the N-glyosidic bond, which was also reported for other bulky C8-arylamine modified nucleosides.78,79
52 N6 amine
H1’
NH 2 chrysene - 8 (6.63, 9.29) N N N N N N H HO O
H OH
Figure 17. ROESY NMR spectra of N-(dA-8-yl)-6-AC adduct (3) in DMSO-d6 at 300 K
To determine the conformational preference of the adducted nucleoside, we attached the 6- aminochyrsene moiety to the template for adenosine in the Gaussian quantum chemistry software and scanned over the O4′−C1′−N9−C4 dihedral in 10° increments.69,80 Consistent with the ROESY
NRM data, we found that the syn conformation is the lowest energy conformer, and that the lowest energy anti conformer is significantly higher in energy (Figure 18). Interestingly, both are stabilized by an intramolecular hydrogen bond. At still considerably higher energy, there is a mix of syn and anti-conformers. These results qualitatively confirms the assignment of the syn conformation preference from experiment, which is also in agreement with similar adducted nucleosides.46,81
53 Level of Theory: B3LYP/6-31G(d)
Figure 18. Potential energy surface scan around the deoxyribose-nucleobase bond of N-(dA-8-yl)- 6-AC adduct (3)
Circular Dichroism (CD) Studies
The 15mer (5’-GCCCTCAACAAGATG-3’) unmodified and N-(dA-8-yl)-6-AC modified oligodeoxynucleotides (5’-GCCCTCAA*CAAGATG-3’) were allowed to anneal to their complementary strand, and the resultant duplexes were evaluated on a circular dichroism spectrometer (ChirascanTM V100). As shown in Figure 19 (Panel A), the CD spectra confirm overall B type DNA conformation of the unmodified control duplex and the N-(dA-8-yl)-6-AC containing duplex. For control duplex a positive Cotton Effect was observed at 280 nm with
54 negative one at 245 nm typically found in B type DNA. However, CD curve of the N-(dA-8-yl)-
6-AC containing
A 3 B 3 Unmodified 15mer Duplex Unmodified 12mer Duplex N-(dA-8-yl)-6-AC 15mer Duplex N-(dA-8-yl)-6-AC 12mer Duplex 2 N-(dA-8-yl)-6-AC in 15mer ssDNA N-(dA-8-yl)-6-AC in 12mer ssDNA 2
1
⟶ 1 ⟶ θ]) θ]) ([
0 ([ 220 260 300 340 380 0 wavelength (nm) ⟶ 220 260 300 340 380 -1 wavelength (nm) ⟶ -1 molarellipticity -2 molarellipticity
-2 -3
-4 -3
Figure 19. CD curves of unmodified (red solid line) and modified (black solid line) 15-mer (Panel
A) and 12-mer (Panel B) oligonucleotides (sequence shown in Table 2) after annealing with their complementary strand. The CD spectra of the single-stranded oligonucleotides are shown in black dotted lines.
Similarly, the 12mer (5’-GTGCATGTTTGT-3’) unmodified and N-(dA-8-yl)-6-AC modified oligodeoxynucleotides (5’-GTGCA*TGTTTGT-3’) were allowed to anneal to their complementary strand, and the resultant duplexes were evaluated on a circular dichroism spectrometer. Likewise, the positive and negative Cotton Effect consistent with canonical B-type
DNA helical structure were observed for 12mer control (unmodified) and N-(dA-8-yl)-6-AC modified duplex. However, both 12mer unmodified and modified duplexes showed less structured helices than that of the 15mer duplexes.
55 Study of the Stability of N-(dA-8-yl)-6-AC Adducted Nucleoside
N-(dA-8-yl)-6-AC DNA adduct is believed to give rise to the corresponding 2'-deoxyinosine adduct, but whether the process of deamination is enzymatic or non-enzymatic has never been established.14,13 We investigated the stability of N-(dA-8-yl)-6-AC adducted nucleoside under biologically relevant conditions; in aqueous solution of PBS buffer at nearly neutral pH at 37 °C.
N-(dA-8-yl)-6-AC adducted nucleoside was incubated in 50 mm phosphate-buffered saline (PBS), pH 7.3 containing 30% v/v DMSO, which was necessary to enhance the solubility. As shown in
Figure 20, N-(dA-8-yl)-6-AC adducted nucleoside had an absorption maximum at λ = 268 nm.
During incubation aliquots were taken at various time points, diluted with water to a final concentration of 0.3 mM, and analyzed by their UV/Vis absorption spectra. The absorption maxima remained constant over 180 hours suggesting that N-(dA-8-yl)-6-AC adducted nucleoside is stable under the experimental conditions. Samples from the UV/Vis experiments were injected into the HPLC to determine the presence of new peaks. However, only a single peak corresponding to N-(dA-8-yl)-6-AC adducted nucleoside standard was observed. Thus, our biochemical in vitro assay suggests that N-(dA-8-yl)-6-AC is stable in aqueous solution at a pH of 7.3, which is the pH detected inside the nucleus and cytoplasm of healthy cells.82 N-(dA-8-yl)-6-AC adducted nucleoside does not undergo spontaneous deamination to deoxyinosine derivative under the conditions described. However, further studies need to be performed in order to unequivocally confirm the stability of N-(dA-8-yl)-6-AC.
56 A 0.9 B 1 0.8 0 min 3 hrs 0.7 12 hrs 0.8 0.6 24 hrs 48 hrs 0.5 72 hrs 0.6 180 hrs 0.4 0.4 0.3 Intensity of Absorption Intensityof 0.2
Intensity of Absorption268at Intensityof 0.2 0.1
0 0 200 300 400 500 600 0 50 100 150 λ (nm) Incubation Time (h)
Figure 20. A) Overlay of the absorption spectra of N-(dA-8-yl)-6-AC adducted nucleoside measured at the indicated time points; incubation of the compound (6 mM) in water at 37°C; for each measurement aliquots were taken and diluted with water to a final concentration of 0.3 mM
B) Plotted data taken from the absorption measurement: intensity of absorption at λ = 268 nm against incubation time.
57 3. 4 Conclusion
Using ROESY NMR spectroscopy, we determined that the adducted purine prefers syn conformation of its glyosidic bond of N-(dA-8-yl)-6-AC in solution. The CD spectra of N-(dA-8- yl)-6-AC 15-mer and 12-mer containing duplexes were consistent with B-DNA, although N-(dA-
8-yl)-6-AC modified duplexes showed small deviations from the control duplexes, suggesting less structured helices for both 12 and 15-mer. In addition, we determined that N-(dA-8-yl)-6-AC nucleoside is stable under in vitro biologically relevant conditions.
58
Chapter 4
Translesion Synthesis and DNA Repair of 6-Nitrochrysene-Derived C8-2′-Deoxyadenosine Adduct in E. coli and Human Cells
†Powell, B.V.;Basu, A.K. “Translesion Synthesis and DNA Repair of 6-Nitrochrysene Derived C8-2’- Deoxyadenosine Adduct in Human Cells.” [Manuscript in preparation]
59 4. Abstract
DNA lesions resulting from 6-NC modifications can inhibit DNA replication and are likely to induce mutations if they are not removed by cellular defense pathways prior to DNA replication.
A previous in vitro study in HeLa cell extracts have shown that the C8-2'-deoxyadenosine DNA adduct derived from 6‑nitrochrysene is more resistant to nucleotide excision repair (NER) than the corresponding C8-2'-deoxyguanosine DNA adduct. In this study, we investigated the replication bypass efficiencies and the roles of translesion synthesis (TLS) DNA polymerases in bypassing the 6NC modified C8-2'-deoxyadenosine adduct, N-(dA-8-yl)-6-AC in human embryonic kidney cells (HEK293T). We further investigated the mutagenicity and translesion synthesis (TLS) efficiency of N-(dA-8-yl)-6-AC in E. coli to compare mutation signature in E. coli with mutations induced in human cells. Replication of plasmid containing a single site-specific N-(dA-8-yl)-6-AC adduct in HEK293T provided ~12 % progeny plasmid with mutations largely A→T transversions.
We observed 4-fold decrease in mutation frequency when the plasmid is replicated in polymerases
κ or ζ HEK293T KO cells. Similar experiments in polymerase η KO cells showed a 1.5-fold increase in mutation frequency and a 38% decrease in TLS bypass efficiency. We also noted that plasmid replication in polymerases ζ KO and κ siRNA KD cells resulted in a 29% decrease in TLS bypass efficiencies and 10-fold decrease in mutation frequency. Taken together, these results suggest that polymerases κ and ζ are involved in the error-prone TLS of N-(dA-8-yl)-6-AC, while pol η is performs error-free bypass. In Escherichia coli, viability of the adducted construct was
~60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the adduct was 5.2%. With SOS, the targeted MF increased 3-fold to 9.0%. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells.
60 Chapter 4 – Introduction
4. 1 Introduction
Ubiquitous environmental contaminants such as nitropolycyclic aromatic hydrocarbons
(NO2-PAH) are known to induce mammary cancer in rats and must therefore be regarded as
7,16,83 potential human carcinogens. A representative example of NO2-PAH is 6-nitrochrysene (6-
NC). It is now clear that 6-NC is activated in cells not only from simple nitroreduction but also from a combination of ring oxidation and nitroreduction.13,14
OH OH
O O NH2 N N N NH NH N HN HN HN N N NH2 N N NH2 N N O O O O O O
O O O
N-(dG-8-yl)-1,2-DHD-6-AC N-(dG-8-yl)-6-AC N-(dA-8-yl)-6-AC
Figure 21. Nucleotide excision repair of 6-NC DNA adducts in HeLa cell extracts.14
61 El-Bayoumy and coworkers evaluated the nucleotides excision repair (NER) propensity of
DNA adducts that is form from 6-NC in an in vitro study. They demonstrated that the N-(dG-8- yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of∼2 and that the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is
∼8- fold lower than that of the N-(dG-8-yl)-6-AC adduct (Figure 20). Thus, the 6-NC-derived adenine adduct represents an example of the resistance of bulky DNA adducts to the human nucleotide excision repair system.14 This suggests that although N-(dA-8-yl)-6-AC adduct is formed in less abundance, it is more persistent in human cells. As a result, it could be potentially more deleterious and mutagenic than the other more abundant DNA damages. DNA adducts that are resistant to NER are persistent in cells can stall replication machinery and might induce mutation at higher frequencies.
For over three decades it has been known that 6-NC is potent carcinogen in laboratory animals.13 However, little is known about the types and frequencies9,20 with which 6-NC induces mutations or the role of TLS enzymes in bypassing DNA lesions that derive from 6-NC in cells.
62 4. 2 Materials and Methods
4. 2. 1 Materials
Chemicals, enzymes, plasmid DNA and cells
All chemicals and reagents and enzymes were purchased from commercial suppliers and used without purification, unless stated otherwise. Chemicals for gel electrophoresis such as agarose, Tris base, boric acid, Na2EDTA, urea, acrylamide, N,N’-methylene bisacrylamide,
TEMED, APS), and preparation of biological media (e.g., agar, yeast extract, bactotryptone, sodium chloride) were purchased from Fisher Scientific (Agawam, MA). Phenol (UltrapureTM
Buffer-Saturated Phenol) for phenol-chloroform extraction was obtained from Invitrogen.
Hybridization buffer (PerfectHybTM Hybridization Buffer 1X) chloroform and isoamyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO). [γ-32P] ATP for gel assays and hybridization were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA).
The media required for mammalian cell culture such as fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), non-essential amino acids (NEAA), penicillin/streptomycin
(PS), Dulbecco’s phosphate buffer saline (D-PBS) solution, 0.25% trypsin-EDTA, Opti-MEM I
TM Reduced Serum Medium and transfection reagent (Lipofectamine 2000) were purchased from
Invitrogen Corp. (Carlsbad, CA).
All enzymes needed for construct preparation and radiolabeling including EcoRV, EcoRI T4
DNA ligase, T4 polynucleotide kinase (PNK), uracil DNA glycosylase (UDG), and exonuclease
III (exo III) were obtained from New England Biolabs (Beverly, MA).
pMS2 plasmid was a gift from M. Moriya (SUNY, Stony Brook, NY).84
63 Escherichia coli strain DH10B and DH12S were purchased from Invitrogen (Carlsbad, CA).
HEK 293T cells were obtained from ATCC (Manassas, VA).
The E. coli strains used were AB1157 [F- thr-1 araC14 leuB6(Am) ∆(gpt-proA)62lacY1 tsx-
33 supE44(AS) galK2(Oc) hisG4(Oc) rfbD1 mgl51 rpoS396(Am) rpsL31(Strr) kdgK51 xylA5 mtl-
1 argE3(Oc) thi-1] were provided by G. Walker (MIT, Cambridge, MA).
The unmodified oligodeoxynucleotides were purchased from Integrated DNA Technology
(Coralville, IA) and Midland Certified Reagent Company (Midland, TX). The modified oligonucleotides were prepared as reported. Oligonucleotides were desalted using a Sep-Pak C18 cartridge from Waters Corp. (Milford, MA).
Synthetic siRNA duplex against POLK (SI04930884), POLI (SI03033310) was purchased from Qiagen (Valencia, CA)
4. 2. 2 Methods Agarose gel electrophoresis for single-stranded vector analysis
Agarose gel electrophoresis was performed to analyze purity of the plasmid preparations and to determine the construct concentration. The ss-pMS2 vector analysis was performed in 1.1% agarose gels. The agarose gel (1.1% w/v, 0.66 mg agarose in 60 mL of 1x TBE) was casted on a horizontal gel apparatus and gel was allowed to solidify for 30 min. The sample was prepared by mixing 0.2 μg (2 μL) of the plasmid DNA with 2 μL of 6x loading dye (10mM Tris-HCl (pH7.6),
0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA) diluted to 10
μL with water. ss-pMS2 analysis gel was run at 110 V for 3 hours at room temperature. Following electrophoresis, the gel was stained with ethidium bromide (25 μg/μL in 500 mL water) for five minutes, de-stained for 30 min in 1x TBE, and the DNA bands were visualized by UV-light.
64 Bacterial electro-competent cell preparation and SOS induction
Four milliliters of overnight E. coli culture, prepared from a single colony, was transferred to 200 mL of 1x LB media. E. coli cells were grown to 1 × 108 cells/mL (~ 2 h) and then harvested by centrifugation at 5000g for 15 min at 0 oC. This procedure was repeated twice except the cells were resuspended in 40 mL ice-cold deionized water. The bacterial pellet was resuspended in 1 mL of glycerol/water (10% v/v) and kept on ice until further use. For SOS response, the following additional steps were introduced after the first centrifugation. The cells were resuspended in 50
2 2 mL 10 mM MgSO4 and treated with 50 J/m or 20 J/m of UV light (254 nm) in 25 mL aliquots in 150 × 50 mm plastic petri dishes. The cultures were incubated in Luria broth at 37 oC for 40 min in order to express SOS functions maximally. Following SOS induction, these cells were centrifuged, deionized, and resuspended in glycerol/water in a similar manner as described earlier except all manipulations were carried out in subdued light.
Large scale preparation of single-stranded pMS2 (sspMS2) vector
Single-stranded phagemid pMS2 DNA was prepared from E. coli JM109 with the aid of the helper phage M13K07 (NEB, Beverly, MA) as reported by Moriya.84 The pMS2 vector contains origins of replication for f1, ColE1, and SV40, which can be used for single stranded replication in E. coli, double stranded replication in E. coli and mammalian cells. It also contains neomycin- resistant (neo) and ampicillin-resistant (amp) genes, which can be used for the selection of the cells containing the vector in mammalian and bacterial cells, respectively. In addition, it has the SV40 early promoter, SV40 small tumor (T) antigen splice sites, SV40 early polyadenylylation sites, and a multiple cloning site with a hairpin loop, that allow digestion of the single-stranded vector with
EcoRV
65 Electrocompetent E. coli DH12S (Invitrogen) cells were thawed on ice and 50 μL of cells were mixed with 50 ng of double stranded replicative form (RF) of pMS2. Then this mixture was transferred to a pre-chilled 1-mm gapped electroporation cuvette and the electroporation was carried out under following conditions: 1.8 kV, 25 μF and 200 Ω. Immediately after electroporation
1 mL of pre-warmed (37 °C) SOC medium (Invitrogen) was added, transferred to 14 mL culture tube and was grown at 37 °C in an orbital shaker at 225-250 rpm for 1 hour. From the resulting culture, 5.0 μL aliquot was plated on lx YT + ampicillin (100μg/ml) and incubated overnight at 37
°C. The following day, about 100 colonies were scraped from the 1x YT plate using a sterile loop, inoculated to 2 L of ampicillin containing 2xYT media along with 1 mL of M13K07 helper phage
(Invitrogen) and incubated at 37 °C. After two hours of growth, 100μg/mL kanamycin was added and the culture was incubated overnight for another 16 hours.
66 grow inoculate culture colonies
dspMS2 1x YT agar plate E. Coli DH12S
centrifuge discard pellet centrifuge
(cell debris)
PEG 8000 + NaCl 2x YT + M13KO7 helper phage + Kanamycin
suspend pellet Extractions Purification Proteinase K 10 % SDS ssPMS2
Figure 22. Workflow for the large-scale preparation of ssPMS2 plasmid.
The phage-infected culture was transferred to 250 mL centrifuge bottles, spun down at
10,000 rcf (20 min, 15°C), the resulting supernatant was transferred to another 250 mL centrifuge bottle and centrifuged at the same condition. Supernatant from the second spin was transferred 2L flask containing 4% (w/v) polyethylene glycol (PEG) and 3.5 M NaCl. The mixture was stirred in ice for at least one hour to precipitate the phage particles. The resulting cloudy solution was centrifuged at 10,000 rcf (20 min, 4 °C) and the pellet obtained was re- suspended in the minimum volume (6 mL) of 1x Tris-EDTA (pH 7.6) buffer. Then the phage particles were re-precipitated with ice-cold 4% (w/v) PEG and 4 M NaCl for an hour and centrifuged at 10,000 rcf (20 min,
4°C). The pallet obtained was re-suspended in 2.4mL of freshly prepared 1x Tris-EDTA (pH 7.6)
67 buffer and centrifuged for 10 min at 4°C and 10000 rpm. The resulting supernatant containing the phage particles was incubated with 0.625 μg/mL proteinase K (Invitrogen) for 16 hours and single- stranded pMS2 was purified from the digested phage solution by a series of phenol: chloroform extractions, followed by ethanol precipitation. Finally, sspMS2 was re-suspended in 200 μL sterile deionized water and quantified on the UV spectrometer. After purification, the purity of the ss- pMS2 was determined by the absorbance ratio of 260 nm to 280 nm followed by analysis on 1.1
% agarose gel.
Single-Stranded pMS2 modified vector construction
O O P -GCCCTCA*CAAGATG-3’ O EcoRV site 3’ 5’
EcoRV Anneal pDNA Gapped Duplex Cut Scaffold
Ligation
3’ 5’ GCCCTCA*CAAGATG GCCCTCA*CAAGATG
Remove Modified Vector Scaffold
Figure 23. Workflow for construction of modified recombinant vector.
The construction of modified pMS2 vectors containing site-specifically incorporated adducts were performed following the method developed by Moriya. For construction of the pMS2 with modified or unmodified 12mer insert, l00 μg (58 pmols) of sspMS2 DNA was digested with 800
68 units of EcoRV at 37 °C for 3 h followed by room temperature overnight (16h). An aliquot of the reaction mixture was run on an l% agarose gel to confirm the complete linearization of the circular vector. An equimolar ratio of a 58-mer scaffold containing deoxyuridinies in place of deoxythymidines was annealed to linear ssDNA in 50 mM NaCl by heating at 90 °C for 5 min followed by slow cooling to 9 °C over 16 h. Annealing of the scaffold generated a gapped duplex that contained a 12mer-gap with complementary sequence of the lesion to be incorporated. An aliquot of the gapped duplex was run on 1% agarose gel with a known amount of control plasmid
(ss-pMS2). The amount of the gapped duplex was determined by comparing the net-intensity of the gapped duplex band to the net-intensity of the known control plasmid using the KODAK
Digital ScienceTM-1D program. After determining the gapped duplex concentration, a 20-fold molar excess of the modified or unmodified 5'-phosphorylated 12mer insert was added and ligated at 16°C overnight with 2800 units of T4 DNA ligase. Unligated oligonucleotides were removed by being passed through Centricon 100 apparatus. Any residual uncut-circular or religated DNA was removed by additional round of EcoRV digestion. The scaffold was removed by enzymatic digestion with uracil DNA glycosylase (UDG, 05 units/μl) and exonuclease-III (0.5 units/μL) at
16°C overnight. The resulting constructs were extracted with phenol: chloroform and precipitated with ethanol. The final constructs were resuspended in deionized distilled-water and were run on
1% agarose gel. The amount of construct was quantified in a manner similar to the way gapped duplex was quantified.
69
Figure 24. Agarose gel analysis for recombinant DNA construction Replication of modified vector in HEK293T cells
The HEK293T wild type and specific polymerase knock out (KO) cells were maintained in
Dulbecco’s modified Eagle’s medium supplemented with 4 mM L-glutamine, and adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10% fetal bovine serum in. When it was about ~80% confluent, cells were washed with PBS (Invitrogen), harvested with 6 mL of Accutase and was seeded in 24-well plate (approximately 1 x 105 cells). The cells were grown to ~80% confluency in the 24 well plate and transfected with 100 ng of each construct using Lipofectamine cationic lipid reagent (Invitrogen) according to manufacturer’s instruction. Subsequent to transfection with control or lesion containing pMS2, the cells were allowed to grow at 37oC, 5%
CO2 for 24 hours and the plasmid DNA was harvested using Qiagen DNA extraction kit.
TLS Assay in Human Cells
The lesion-containing or control pMS2 vector was mixed with a single-stranded pMS2 DNA vector (internal control); a 23-mer oligodeoxynucleotide sequence different from the N-(dA-8-yl)-
70 6-A containing sequence (or control) DNA sequence. The molar ratio of internal control vector to control vector was 1:1, and those for the internal control vector to N-(dA-8-yl)-6-AC containing vector was 1:3. The a 23-mer oligodeoxynucleotide vector was used as an internal control and gave equal number of progeny as the control vector. TLS efficiency was determined as the percentages of the colonies originating from the N-(dA-8-yl)-6-A containing plasmid relative to the internal control.
Mutant screening by oligonucleotide hybridization
Dot- blot hybridization was performed to identify the possible mutations at the lesion- site or at nearby locations in the progeny. In brief, appropriate amounts of liquid culture were plated on lx
YT + ampicillin (100μg/mL) plates and incubated at 37 °C overnight (16-18 h). The resulting individual colonies were picked using sterile toothpicks into single wells of a 96-well plate (Fisher
Scientific) containing 200 μl lx YT + ampicillin (100 μg/mL). The inoculated 96- well plate was then incubated at 37°C for a minimum of 3 hrs before blotting colonies onto filter paper. Using a
96-well replicator (Boekel Scientific, Feasterville, PA), cultures in the 96-well plate were blotted onto sterile labeled Whatmann chromatography papers placed on lx YT + ampicillin (100 μg/mL) plates, in triplicates. The plates were incubated at 37 °C for 16 h for colonies to be replication on the filter paper. After overnight incubation, following procedure was carried out to fix DNA onto the filter paper. The filters were carefully lifted from the plates and subsequently transferred to
(colony side up) 0.5 N NaOH, denaturing solution, for 12 mins, and to 0.5 M Tris-HCl (pH 7.5), neutralizing solution for 7 mins. Then the filters were washed twice with lx SSC for 4 mins with slow agitation (50 rpm) and once with 100% ethanol for 30 seconds before they were baked for 2 hours at 80 °C. In-between each washing steps, except for two SSC washes, filters were drained and damped dry on paper towels (colony side up). Thereafter, each filter was placed separately in
71 a heat sealable bag and sealed with 6 mL of PerfectHybeTM hybridization buffer (Sigma) containing corresponding left, right or wild-type 32P-radiolabeled probe (10 pmol of probe/filter).
Liquid in the sealed bags were spread evenly to entirely cover the filters and were allowed to hybridize to corresponding probe at its optimum hybridization temperature for a minimum of 4 hours in the hybridization oven (ProblotTM 12). Then the filters were carefully removed from the bags and gently agitated (60 rpm) twice with 2x SSC+0.1% SDS, preheated to the hybridization temperature (THyb), for 10 mins. Finally, the filters were removed from the SSC solution, dried and exposed to autoradiography. Two left and right probes were used to select phages containing the correct insert, and transformants that did not hybridize with both the left and right probes were omitted. Any transformants that hybridized with the left and right probes but failed to hybridize with the wild-type probes were subjected to DNA sequence analysis.
Transformation of modified vector in E. coli and progeny analysis
Competent E. coli cells were prepared as described earlier. To induce SOS, the cells were re- suspended in 10 mM MgSO4 solution and aliquots were treated with UV light (254 nm) (20 J/m2) in plastic Petri dishes. The combined aliquots were incubated in Luria broth at 37° C for 40 min for maximum SOS induction. Following SOS induction, these cells were processed in a similar manner, except the procedure was carried out in subdued light. Then transformation of cells was carried out in a Bio-Rad Gene-Pulser apparatus by mixing vector DNA and un-induced cells or
SOS-induced cells. Cells were grown for 1 h at 37° C after adding SOC medium and then plated on a 1X YT plates containing Ampicillin and the transformants were analyzed by hybridization.
72 4. 3 Results and Discussion
The strategy for construction of the adduct containing single stranded vector was developed by Moriya84 and has used been successfully by our lab85–88 and many other researchers.89–91 We used pMS2 shuttle vector plasmid containing the origins of replication for f1, ColE1, and SV40, and the genes for neo and amp resistance.
O O P -GCCCTCA*CAAGATG-3’ O EcoRV site 3’ 5’ GCCCTCA*CAAGATG 1. EcoRV 1. Ligation pDNA Gapped Duplex Modified Vector 2. Anneal 2. Scaffold scaffold removal
1. Replication (HEK293T) 2. Plasmid recovery X 1. Blot transfer 1.Transform in GCCCTCA*CAAGATG to filter (E. coli)
2. Grow for 16h 2. Grow on 3. Hybridization ampicillin plates with radiolabel probes Progeny Phagemid
Oligonucletide Hybridization
Left Probe Wild Type Probe Right Probe
DNA Sequencing
Figure 25. A general scheme for construction of the lesion-containing vector and replication in E. coli or HEK 293T cells, and analysis of the progeny
73 This single-stranded pMS2 vector also contain a hairpin region that, upon digestion with the
EcoRV restriction enzyme followed by scaffolding with an oligonucleotide, generates the desired gapped duplex (Figure 25). The 15-mer oligodeoxynucleotide (5’-GCCCTCAA*CAAGATG-3’) containing N-(dA-8-yl)-6-AC adduct is ligated to this gap and the scaffold was enzymatically removed before replication. The strategy for construction and mutant screen assay is shown in
Figure 25.
Mutational analyses of N-(dA-8-yl)-6-AC in E. coli
The DNA sequence of the 15-mer was chosen from TP53 gene codon 129-133, because crops contaminated with another nitroaromatic carcinogen aristolochic acid (AA) causes A→T mutation in codon 131 in patients with urothelial tumors. While many polyaromatic amine and nitroaromatic compounds form adducts at the C8 position of dG, which induce frameshifts and G→T transversions as the dominant mutations, the major adducts by AA are formed at the N6 position of dA. The N-(dA-8-yl)-6-AC containing plasmid was replicated in uninduced and SOS-induced
E. coli AB1157 cells. In uninduced cells, the number of progeny colonies from the adduct containing construct was reduced to ~60% to that of the control (Figure 26A). This suggests that though replication was inhibited, N-(dA-8-yl)-6-AC is less toxic than the C8 dG adduct of 1- nitropyrene,8,78 which gives less than 30% progeny. Upon induction of SOS, viability of the adducted genome increased to nearly 90%, indicating significantly more facile translesion synthesis (TLS) of N-(dA-8-yl)-6-AC by the SOS proteins (Figure 26A).
Without SOS, 5.2% of the replicates were mutants, which comprised of 2.9% targeted and
2.3% semi-targeted mutants (Figure 26B). Of the targeted mutations, 80% was A*→G transitions.
With SOS, overall MF increased to 12.2%, showing three-fold increase of the targeted mutations to 9% frequency, whereas semi-targeted mutation increased only marginally to 3.2% (Figure 26C).
74 Although A*→G was also the prevalent (55%) targeted mutation with SOS, nearly half as many
A*→C was detected.
A B C
12 120 Targeted Control Semi-targeted 7 (+) SOS N-(dA-8-yl)-6-AC *** 10 100 6 * *
80 8 5
4 60 6 3 (-) SOS * * 40 4 2 TLS Efficiency (%) Efficiency TLS MutationFrequency %
20 2 MutationFrequency % 1
0 0 0 (-) SOS (+) SOS (-) SOS (+) SOS A*→T A*del A*→T A*→G A*→GA*→C
Figure 26. (A) The bypass efficiencies in N-(dA-8-yl)-6-AC construct in E. coli (-) SOS (WT) and
(+) SOS cells. The data represent the means and standard errors of the mean of results from 3 independent replication experiments. (B) The frequencies of total targeted mutations (C) induced in progeny from N-(dA-8-yl)-6-AC construct in (-) SOS (WT) and (+) SOS E. coli cells. The data represent the means and standard errors of the mean of results from 3 independent replication experiments. *P < 0.05; ***P < 0.001. The P-values were calculated by using unpaired two-tailed
Student’s t-test.
75 Translesion Synthesis of N-(dA-8-yl)-6-AC in Human Cells
To investigate the replication properties of N-(dA-8-yl)-6-AC adduct in human cells and the role of TLS polymerases in bypassing this lesion in cells; we conducted replication experiments in HEK293T cells and in HEK293T isogenic cells where individual TLS DNA polymerases were depleted by the CRISPR/Cas9 genome editing method and siRNA knock down approach.79,92 To study the role of specific TLS polymerase in the bypass of a lesion many researchers have conducted replication experiments in cells where specific TLS polymerase have been knockdown by siRNA approach.85,86 However, the siRNA method often results in incomplete depletion of TLS polymerases. The presence of residual amounts of TLS Pols makes is difficult to draw clear conclusions about the role of specific polymerases in lesion bypass.92,93 The recent advances in
CRISPR-Cas9 genome editing method has enabled the facile knockout of individual genes in cultured human cells .94,95 To determine the TLS efficiency of N-(dA-8-yl)-6-AC adduct in HEK
293T cells, we mixed 3:1 ratio of N-(dA-8-yl)-6-AC vector and unmodified plasmid that contained a different unmodified sequence. The unmodified plasmid was used as an internal control. The percentages of the colonies originating from the N-(dA-8-yl)-6-AC lesion-containing construct relative to the unmodified plasmid, reflecting the percentage of TLS, were determined by oligonucleotide hybridization.
In contrast to TLS efficiency in E. coli WT cells which was 60%; TLS replication efficiency was 83% in HEK293T WT cells in which all TLS polymerases are expressed (Figure 27). The largest decrease in TLS efficiency was observed in Pol η KO cells (45%). In Pol κ and Pol ζ KO cell TLS efficiencies were ~60 % and ~70 % respectively. TLS efficiency decreased only marginally in Pol ι KO cells. TLS efficiencies in Pol η/κ and Pol η /ζ double KO cells were ~25 % and ~35% respectively. In Pol ζ KO and Pol κ KD cells TLS efficiency was ~54%. On the basis
76 of TLS replication bypass efficiencies, the role of TLS Pols in the bypass of N-(dA-8-yl)-6-AC can be rank as follows Pol η > Pol κ > Pol ζ > Pol ι.
TLS Bypass Adduct Signals X 100% Efficiency = Control Signals 90
80 * 70
*** 60 **
50 ***
40 ***
30 ***
20 TLS Bypass Efficiency (%) 10
0 - - ι- ζ- - - - WT η κ η/ζ Pol Pol Pol Pol η/κ ζ/κ Pol Pol Pol
Figure 27. TLS bypass efficiencies of the N-(dA-8-yl)-6-AC containing construct in HEK293T cells and various TLS polymerase KO HEK 293T cells. The data represent the means and standard
77 errors of the mean from 2-4 independent replication experiments. *P < 0.05; **P < 0.01; ***P <
0.001. The P-values were calculated by using unpaired two-tailed Student’s t-test.
Mutational analyses of N-(dA-8-yl)-6-AC in Human Cells
DNA sequence analysis showed that N-(dA-8-yl)-6-AC is mutagenic in HEK293T cells.
Replication of plasmid containing a single site-specific N-(dA-8-yl)-6-AC adduct in HEK293T wild type cells provided ~12 % progeny plasmid with mutations that included ~11% at the targeted site and ~ 1% occurred at the 3’ or 5’ end of the target site and are referred to as semi-targeted mutations (Figure 28). In HEK293T wild type cells, mutations at the target site that were largely
A→T transversions. We observed 4-fold decrease in mutation frequency when the plasmid is replicated in polymerases κ or ζ HEK293T individually KO cells. Similar experiments in polymerase η KO cells showed a 1.5-fold increase in mutation frequency. We also noted that plasmid replication in polymerases ζ KO and κ siRNA knock down cells resulted in 10-fold decrease in mutation frequency.
78 16 Targeted ** 14 Semi-targeted
12
10 **
8
6
** ** ** 4 Mutation Frequency % **
2 ***
0 WT Pol η- Pol κ- Pol ι- Pol ζ- Pol η/ζ- Pol η/κ- Pol ζ/κ-
Figure 28. Targeted and semi-targeted mutations induced in the progeny from the N-(dA-8-yl)-6-
AC construct in HEK 293T and various polymerase knockout cells. The data represent the mean and the standard deviation (of the total MF) from 2-4 independent experiments. The statistical significance of the difference in MFs between HEK 293T and TLS pols knockouts was calculated using two-tailed, unpaired Student’s t test. (*P < 0.05; **P < 0.01; ***P < 0.001).
79 1010 * A*→T A*→T A*→G A*→GA*→C 88 A*→C
66 ** * * 44 * *
22 **
Mutation Frequency % * * *
Mutation Frequency% Mutation ** ** 00 WTWT PolPol ηη-- PolPol κκ-- Pol ιι-- Pol ζ- Pol η/ζ- Pol η/κ- Pol κ /ζ-
Figure 29. The types and frequencies of targeted mutations induced in the progeny from the N-
(dA-8-yl)-6-AC construct in HEK 293T cells and various polymerase knockout HEK293T cells.
The data represent the mean and the standard deviation (of the total targeted MF) from 2-4 independent experiments. The statistical significance of the difference in targeted MFs between
HEK 293T and TLS pols knockouts was calculated using two-tailed, unpaired Student’s t test. (*P
< 0.05; **p < 0.005).
The Role of Specific TLS Polymerases in N-(dA-8-yl)-6-AC TLS Bypass
The results from TLS replication efficiency assay and DNA sequencing analysis (Figure 29) suggest that Pol η is strongly involved in bypassing N-(dA-8-yl)-6-AC DNA lesion in an error free manner. Neither TLS bypass efficiency nor mutation frequency differed significant from the wild type results when N-(dA-8-yl)-6-AC vector was replicated Pol ι KO cells. This suggests that Pol ι does not play a significant role in bypass of N-(dA-8-yl)-6-AC DNA lesion. We observed 4-fold decrease in mutation frequency when the plasmid is replicated in polymerases κ or ζ HEK293T
KO cells. A*→ T transversions were the main mutation induced by N-(dA-8-yl)-6-AC adduct in
80 HEK293T cell. However, in Pol κ KO cells A*→ G transitions were induced at a higher frequency than A*→ T transversions. This suggests that Pol κ in responsible for inducing a significant amount of A*→ T transversions when N-(dA-8-yl)-6-AC vector is replicated in HEK293T cells.
Furthermore, the decrease in mutation frequency in the absence of polymerases κ and ζ suggests that polymerases κ and ζ are strongly involve in the error-prone or mutagenic bypass of N-(dA-8- yl)-6-AC. Replication experiments in cells where κ and ζ are simultaneously depleted resulted in
10 fold decrease in mutation frequency suggesting that κ and ζ are cooperatively involved in the error-prone bypass of N-(dA-8-yl)-6-AC adduct.
81 4. 4 Conclusion
In conclusion, N-(dA-8-yl)-6-AC is mutagenic in E. coli and HEK293T cells. In E. coli, N-
(dA-8-yl)-6-AC DNA adduct induces ~5 % mutant progeny in wild type cells. The induction of
SOS response increases lesion bypass efficiency from ~60 % (wild type cells) to ~90% in SOS induced cells and increased targeted mutation in progeny 3-fold. This can be attributed to increase expression of low fidelity TLS polymerases pol II, pol IV and pol V. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells. In contrast, In HEK293T cells
N-(dA-8-yl)-6-AC DNA adduct induces~12 % progeny plasmid with mutations largely A*→T transversions. In HEK293T cells, polymerases κ and ζ are cooperatively involved in the error- prone bypass of N-(dA-8-yl)-6-AC adduct while pol η in strongly involved in the error-free bypass.
Thus, our study provided important new insight into cellular replication properties of N-(dA-8-yl)-
6-AC lesion and defined the roles of TLS polymerases in bypassing this lesion in E. coli and human cells.
82
Chapter 5
Mutagenicity and Genotoxicity of Fapy.dG DNA Adduct in Human Cells
83 5. Abstract
Fapy·dG are formed in DNA by hydroxyl radical damage. In order to study replication bypass efficiency and mutation outcomes of Fapy.dG lesion in HEK293T cells, we constructed a single- stranded shuttle vector containing the lesion. Two plasmid vectors containing Fapy.dG adduct at the G1- or G3-positions of a part p53 sequence, 5’-AAC CG1G AG3G CCC-3’ containing mutation hotspot codon 248 (red) or codon 249 (green). Replication of vectors in human embryonic kidney
HEK293T cells, showed that Fapy.dG DNA adduct is mutagenic inducing largely G*→T transversions. Furthermore Fapy.dG adduct in CG*G sequence context (codon 248) induce mutations at ~50% greater frequency than AG*G in codon 249. The roles of several translesion synthesis (TLS) replication polymerases in adduct bypass was also investigated via conducting replication experiments in the isogenic cells where individual TLS DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Mutational analyses in HEK293T KO cells are in progress.
84 5. 1 Introduction
Oxidative DNA damage from reactive metabolites produced in cells can play important roles in aging and various diseases.96,97 Ionizing radiation generates many lesions in DNA that are formed by oxidative stress. It is now clear that oxidative DNA damage induces many different lesions in DNA, of these lesions 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) has received the most of attention.98 However, oxidative DNA damages also generates ring-opened formamidopyrimidine derivative, Fapy.dG. It is believed that 8-oxo-dG and Fapy.dG derives from a common radical intermediate (Scheme 8).99–102
O H N NH O N N NH O 2 O
O
OX. 8-oxo-dG
O O
N NH HO N NH HO N N NH H N N NH O 2 O 2 O O
O O H O O Red. HN NH
HN N NH O 2 O
O Fapy.dG Scheme 6. Postulated hydroxyl radical mediated pathway to Fapy.dG and 8-oxo-dG
Fapy·dG DNA lesions is subject to repair by excision repair enzymes. Formamidopyrimidine glycosylase (fpg, MutM) is known to excise several oxidative damages such as Fapy.dG.101 The site-specific synthesis of Fapy.dG DNA adduct and incorporation in oligodeoxynucleotides have been accomplished.103,104 These advances in the synthesis of oligodeoxynucleotides containing
85 Fapy·dG have enabled biological studies that investigate biological endpoints of Fapy.dG in cells and the role TLS polymerases in bypassing Fapy.dG adduct in cells.105 Kalam et. al. showed that that Fapy·dG is mutagenic when the modified vector containing Fapy.dG is replicated in simian kidney (COS-7) cells, inducing primarily targeted Fapy·G→T transversions. In the 5’-TGT sequence mutational frequency of Fapy·dG was ~30%, whereas in the 5’-TGA sequence it was
~8%.87 Using a similar approach, Pande et. al. reported that DNA polymerase λ is responsible for a significant portion of Fapy·dG induced G → T mutations in human cells, whereas it conducts predominantly error-free bypass of 8- oxo-dG.85
H O O HN NH 5’-AAC CG*G AGG CCC-3’ HN N NH 248 G* = O 2 O 5’-AAC CGG AG*G CCC-3’ 249 O Fapy.dG
Figure 30. Oligonucleotide sequences from the p53 hotspots that include codons 248 and 249
In contrast, this work aims at determining the mutational signatures of Fapy.dG in interesting p53 hotspots sequences that include codons 248 and 249. The mutations in p53 gene in cancer are distinct and have been noted in several hot spots such as at codons 157, 175, 248, 249, and 273, which correspond to amino acids within the DNA binding domain of p53.21,106,107 In human breast cancer, p53 codon 248 is most frequently mutated. Nearly 50% of all colon cancers contain mutations at the three CpG hot spots at codons 175, 248, and 273.107 By contrast, in hepatocellular carcinoma, G →T transversion of the second G in codon 249 occurs frequently, in which the mutated G appears in a GpG sequence. Indeed, GpG sequences
108 are hot spots for oxidative damage. Furthermore, oxidative damage of human fibroblasts with H2O2 and
86 109 FeCl3 induced G:C →T:A transversions at p53 codons 249 , which is also a susceptible site in DNA isolated from the liver tissues of Wilson’s disease patients.110
5. 2 Material and Methods
5. 2. 1 Synthesis of Fapy.dG containing oligonucleotides
The synthesis and characterization of Fapy·dG 12mers, 5’-AAC CG*G AGG CCC-3’and 5’-AAC
CGG AG*G CCC-3 were synthesized in the laboratory of Prof. Marc Greenberg and have been reported.36,98,104 The unmodified and complementary oligodeoxynucleotide sequences were purchased from Integrated DNA Technologies, Inc in Coralville, Iowa, USA.
5. 2. 2. Single stranded vector construction and replication studies.
Procedure for construction and characterization of pMS2 vectors containing a single Fapy·dG site are reported in section 4. 2. 2 of thesis.
87 5. 3 Results and Discussion
To investigate the mutation outcome of Fapy.dG in p53 hotspot sequences, single-stranded pMS2 vectors bearing a single Fapy.dG adducted site were constructed with different modified oligodeoxynucleotides. Modified vectors were constructed with 5’-AAC CG*G AGG CCC-3’
* (dG1-Fapy) where codon 248 is underlined and with 5’-AAC CGG AG G CCC-3 (dG3-Fapy) where mutation hotspot codon 249 is underlined. These adduct containing oligodeoxynucleotides and unmodified control oligodeoxynucleotides were inserted into plasmids to construct site- specifically modified vectors using recombinant DNA technology. These vectors were replicated human embryonic kidney (HEK293T) cells. The resulting double stranded plasmid DNA was isolated and analyzed via oligonucleotide hybridization and DNA sequencing (Figure 30). Mutant Screening Workflow
CGGGAGTXGTTCTAC CGGGAGTX*GTTCTAC Grow on GCCCTCAA*CAAGATG 1. Amplified cellulose In E.coli 1. Pick colonies paper
2. Grown on 2. Blot transfer amp. plates
DNA Oligonucleotide Hybridization immobilization Wild Type RP LP CGGGAGTXGTTCTAC CGGGAGTX*GTTCTAC GCCCTCAA*CAAGATG
DNA Sequencing 30
Figure 31. Progeny plasmid processing via oligonucleotide hybridization and DNA sequencing
Oligonucleotide hybridization and DNA sequencing showed that Fapy.dG is mutagenic in
HEK293T cells, inducing largely G*→T transversions. Furthermore Fapy.dG adduct in CG*G sequence context (codon 248) induce mutations at ~50% greater frequency than AG*G in codon
88 249. TLS replication bypass efficiencies of the Fapy.dG containing vector in HEK293T cells were
~90% and ~88% respectively for dG1-Fapy (codon 248) and dG3-Fapy (codon 249) sequences.
Thus, bypass efficiency for Fapy.dG in G1 or G3 sequence were essentially the same in both sequence context.
100 8 dG1-Fapy
95
6 90 dG3-Fapy 85 4
80 TLS Bypass Efficiency TLS Mutation Frequency Mutation 2 75
70 CT dG1-Fapy dG3-Fapy 0
G*→ TG*→ CG*→ A G*→ TG*→ CG*→ A
5’-AAC CG*G AGG CCC-3’ 5’-AAC CGG AG*G CCC-3’ 248 249
dG1-Fapy dG3-Fapy Figure 32. (A) The bypass efficiencies of Fapy.dG constructs (G1 and G3) in HEK293T cells. The data represent the means and standard errors of the mean of results from 3 independent replication experiments. (B) The frequencies of total targeted mutations induced in progeny from Fapy.dG construct in dG1-Fapy and dG3-Fapy.
DNA sequencing analysis showed that Fapy.dG in codon 248 induces ~12% mutant progeny and induces ~5% mutant progeny in codon 249. In both sequences the main mutation is G*→T
89 transversions at the target site. In similar work from our lab, Pande et al. showed that
Fapy.dG induces 10−22% mutations, predominantly G → T transversions, in human embryonic kidney 293T cells in four TG*N sequence contexts, where N = C, G, A, or T.85 This current study aims at investigating and defining the mutation signature of Fapy.dG in p53 mutation hotspots sequences realized from cancer patients.
5. 4 Conclusion
In conclusion, Fapy.dG is mutagenic in HEK293T cells inducing mainly G→T transversions. In the p53 sequence, the lesion is most mutagenic when located at G1 (codon 248).
Nonetheless, Fapy.dG is also mutagenic when located at G3 position (codon 249).
5. 5 Future Direction
The effect of specific TLS replication proteins in bypassing Fapy.dG lesion in codon 248 and 249 p53 sequences will be assessed by performing TLS assay in CRISPR/Cas9 genome edited knockout human embryonic kidney cells (HEK293). TLS efficiency will be assessed with pol η, pol κ, pol ι, Rev 1 single knock out cells; followed by TLS assay in double/ triple knock out cells.
90 References
(1) Hoeijmakers, J. H. J. Genome Maintenance for Preventing Cancer. DNA Repair (Amst). 2001, 411 (May), 366–374. https://doi.org/10.1038/35077232.
(2) Lindahl, T.; Wood, R. D. Quality Control by DNA Repair 10.1126/Science.286.5446.1897. Science (80-. ). 1999, 286 (5446), 1897–1905.
(3) Shrivastav, N.; Li, D.; Essigmann, J. M. Chemical Biology of Mutagenesis and DNA Repair: Cellular Responses to DNA Alkylation. Carcinogenesis 2009, 31 (1), 59–70. https://doi.org/10.1093/carcin/bgp262.
(4) Geacintov, N. E.; Broyde, S. Repair-Resistant DNA Lesions. Chem. Res. Toxicol. 2017, 30 (8), 1517–1548. https://doi.org/10.1021/acs.chemrestox.7b00128.
(5) Valko, M.; Izakovic, M.; Mazur, M.; Rhodes, C. J.; Telser, J. Role of Oxygen Radicals in DNA Damage and Cancer Incidence. Mol. Cell. Biochem. 2004, 266 (1–2), 37–56.
(6) Basu, A. K.; Nohmi, T. Chemically-Induced DNA Damage, Mutagenesis, and Cancer. Int. J. Mol. Sci. 2018, 19 (6), 1–4. https://doi.org/10.3390/ijms19061767.
(7) Hecht, S. S.; El-Bayoumy, K.; Rivenson, A.; Amin, S. Potent Mammary Carcinogenicity in Female CD Rats of a Fjord Region Diol-Epoxide of Benzo[c]Phenanthrene Compared to a Bay Region Diol-Epoxide of Benzo[a]Pyrene. Cancer Res. 1994, 54 (1), 21–24.
(8) Monographs, I.; Evaluation, O. N. T. H. E.; Risks, O. F. C.; Humans, T. O. Diesel and Gasoline Engine Exhausts. IARC Monogr. Eval. Carcinog. Risks Hum. 1989, 46, 41–185.
(9) Chae, Y. H.; Yun, C. H.; Guengerich, F. P.; Kadlubar, F. F.; Bayoumy, K. El. Roles of Human Hepatic and Pulmonary Cytochrome P450 Enzymes in the Metabolism of the Environmental Carcinogen 6-Nitrochrysene. Cancer Res. 1993, 53 (9), 2028–2034.
91 (10) El-Bayoumy, K.; Desai, D.; Boyiri, T.; Rosa, J.; Krzeminski, J.; Sharma, A. K.; Pittman, B.; Amin, S. Comparative Tumorigenicity of the Environmental Pollutant 6-Nitrochrysene and Its Metabolites in the Rat Mammary Gland. Chem. Res. Toxicol. 2002, 15 (7), 972–978. https://doi.org/10.1021/tx020019a.
(11) El-Bayoumy, K. Environmental Carcinogens That May Be Involved in Human Breast Cancer Etiology. Chem. Res. Toxicol. 1992, 5 (5), 585–590. https://doi.org/10.1021/tx00029a001.
(12) Purohit, V.; Basu, A. K. Mutagenicity of Nitroaromatic Compounds. Chem. Res. Toxicol. 2000, 13 (8), 673–692. https://doi.org/10.1021/tx000002x.
(13) Delclos, K. B.; Miller, D. W.; Lay, J. O.; Casciano, D. A.; Walker, R. P.; Fu, P. P.; Kadlubar, F. F. Identification of C8-Modified Deoxyinosine and N2-and C8-Modified Deoxyguanosine as Major Products of the in Vitro Reaction of N-Hydroxy-6- Aminochrysene with DNA and the Formation of These Adducts in Isolated Rat Hepatocytes Treated with 6-Nitrochrysene A. Carcinogenesis 1987, 8 (11), 1703–1709. https://doi.org/10.1093/carcin/8.11.1703.
(14) Krzeminski, J.; Kropachev, K.; Reeves, D.; Kolbanovskiy, A.; Kolbanovskiy, M.; Chen, K. M.; Sharma, A. K.; Geacintov, N.; Amin, S.; El-Bayoumy, K. Adenine-DNA Adduct Derived from the Nitroreduction of 6-Nitrochrysene Is More Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts. Chem. Res. Toxicol. 2013, 26 (11), 1746– 1754. https://doi.org/10.1021/tx400296x.
(15) Imaida, K.; Uneyama, C.; Ogasawara, H.; Hayashi, S.; Fukuhara, K.; Miyata, N.; Takahashi, M. Induction of Colon Adenocarcinomas in CD Rats and Lung Adenomas in ICR Mice by 6-Nitrochrysene: Comparison of Carcinogenicity and Aryl Hydrocarbon Hydroxylase Induction in the Target Organs of Each Species. Cancer Res. 1992, 52 (6), 1542–1545.
(16) Rundle, A. Carcinogen-DNA Adducts as a Biomarker for Cancer Risk. Mutat. Res. -
92 Fundam. Mol. Mech. Mutagen. 2006, 600 (1–2), 23–36. https://doi.org/10.1016/j.mrfmmm.2006.05.031.
(17) Takamura-Enya, T.; Kawanishi, M.; Yagi, T.; Hisamatsu, Y. Structural Identification of DNA Adducts Derived from 3-Nitrobenzanthrone, a Potent Carcinogen Present in the Atmosphere. Chem. - An Asian J. 2007, 2 (9), 1174–1185. https://doi.org/10.1002/asia.200700061.
(18) El-Bayoumy, K.; Sharma, A. K.; Lin, J.-M.; Krzeminski, J.; Boyiri, T.; King, L. C.; Lambert, G.; Padgett, W.; Nesnow, S.; Amin, S. Identification of 5-(Deoxyguanosin-N2- Yl)- 1,2-Dihydroxy-1,2-Dihydro-6-Aminochrysene as the Major DNA Lesion in the Mammary Gland of Rats Treated with the Environmental Pollutant 6-Nitrochrysene. Chem. Res. Toxicol. 2004, 17 (12), 1591–1599. https://doi.org/10.1021/tx049849+.
(19) Fagin, J. A.; Matsuo, K.; Karmakar, A.; Chen, D. L.; Tang, S. H.; Koeffler, H. P. High Prevalence of Mutations of the P53 Gene in Poorly Differentiated Human Thyroid Carcinomas. J. Clin. Invest. 1993, 91 (1), 179–184. https://doi.org/10.1172/JCI116168. (20) Muller, P. A. J.; Vousden, K. H. Mutant P53 in Cancer: New Functions and Therapeutic Opportunities. Cancer Cell 2014, 25 (3), 304–317. https://doi.org/10.1016/j.ccr.2014.01.021.
(21) Hainaut, P.; Soussi, T.; Shomer, B.; Hollstein, M.; Greenblatt, M.; Hovig, E.; Harris, C. C.; Montesano, R. Database of P53 Gene Somatic Mutations in Human Tumors and Cell Lines: Updated Compilation and Future Prospects; 1997; Vol. 25.
(22) Moriya, M.; Slade, N.; Brdar, B.; Medverec, Z.; Tomic, K.; Jelaković, B.; Wu, L.; Truong, S.; Fernandes, A.; Grollman, A. P. TP53 Mutational Signature for Aristolochic Acid: An Environmental Carcinogen. Int. J. Cancer 2011, 129 (6), 1532–1536. https://doi.org/10.1002/ijc.26077.
(23) Vom Brocke, J.; Krais, A.; Whibley, C.; Hollstein, M. C.; Schmeiser, H. H. The
93 Carcinogenic Air Pollutant 3-Nitrobenzanthrone Induces GC to TA Transversion Mutations in Human P53 Sequences. Mutagenesis 2009, 24 (1), 17–23. https://doi.org/10.1093/mutage/gen049.
(24) Poon, L. S.; Huang, N. M.; Choo, Y.; McPherson, J. R.; Yu, W.; Heng, L. H.; Gan, A.; Myint, S. S.; Siew, Y. E.; Ler, D. L.; et al. Mutation Signatures Implicate Aristolochic Acid in Bladder Cancer Development. Genome Med. 2015, 7 (1). https://doi.org/10.1186/s13073- 015-0161-3.
(25) Fujii, S.; Fuchs, R. P. Translesion DNA Synthesis and Mutagenesis in Prokaryotes. Cold Spring Harb. Perspect. Biol. 2013. https://doi.org/10.1038/ncomms3243.
(26) Goodman, M. F.; Woodgate, R. Translesion DNA Polymerases. Cold Spring Harb. Perspect. Biol. 2013, 5 (10), 1–20. https://doi.org/10.1101/cshperspect.a010363.
(27) Lehmann, A. R. Translesion Synthesis in Mammalian Cells. Exp. Cell Res. 2006, 312 (14), 2673–2676. https://doi.org/10.1016/j.yexcr.2006.06.010.
(28) Krokan, H. E.; Bjoras, M. Chapter 06: Base Excision Repair. In DNA Repair and Mutagenesis. American Sociey for Microbiology, Washington, DC; Cold Spring Harbor Laboratory Press, 2008; Vol. 5, pp 1–17. https://doi.org/10.1101/cshperspect.a012583.
(29) Beard, W. A.; Horton, J. K.; Prasad, R.; Wilson, S. H. Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism. Annu. Rev. Biochem. 2019, 88 (1), 137–162. https://doi.org/10.1146/annurev-biochem-013118-111315.
(30) Scharer, O. D. Nucleotide Excision Repair in Eukaryotes. Cold Spring Harb. Perspect. Biol. 2013, 5 (10), a012609–a012609. https://doi.org/10.1101/cshperspect.a012609.
(31) Spivak, G. Nucleotide Excision Repair in Humans. DNA Repair. Elsevier B.V. 2015, pp 13–18. https://doi.org/10.1016/j.dnarep.2015.09.003.
94 (32) Fujii, S.; Fuchs, R. P. Translesion DNA Synthesis and Mutagenesis in Prokaryotes. Cold Spring Harb. Perspect. Biol. 2013, 1–22. https://doi.org/10.1038/ncomms3243.
(33) Waters, L. S.; Minesinger, B. K.; Wiltrout, M. E.; D’Souza, S.; Woodruff, R. V.; Walker, G. C. Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance. Microbiol. Mol. Biol. Rev. 2009, 73 (1), 134–154. https://doi.org/10.1128/MMBR.00034-08.
(34) Lehmann, A. R.; Niimi, A.; Ogi, T.; Brown, S.; Sabbioneda, S.; Wing, J. F.; Kannouche, P. L.; Green, C. M. Translesion Synthesis: Y-Family Polymerases and the Polymerase Switch. DNA Repair (Amst). 2007, 6 (7), 891–899. https://doi.org/10.1016/j.dnarep.2007.02.003.
(35) Limoli, C. L.; Giedzinski, E.; Morgan, W. F.; Cleaver, J. E. Polymerase η Deficiency in the Xeroderma Pigmentosum Variant Uncovers an Overlap between the S Phase Checkpoint and Double-Strand Break Repair. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (14), 7939–7946. https://doi.org/10.1073/pnas.130182897.
(36) Haracska, L.; Yu, S. L.; Johnson, R. E.; Prakash, L.; Prakash, S. Efficient and Accurate Replication in the Presence of 7,8-Dihydro-8-Oxoguanine by DNA Polymerase η. Nat. Genet. 2000, 25 (4), 458–461. https://doi.org/10.1038/78169.
(37) Zhang, Y. Error-Prone Lesion Bypass by Human DNA Polymerase Eta. Nucleic Acids Res. 2000, 28 (23), 4717–4724. https://doi.org/10.1093/nar/28.23.4717.
(38) Albertella, M. R.; Green, C. M.; Lehmann, A. R.; O’Connor, M. J. A Role for Polymerase η in the Cellular Tolerance to Cisplatin-Induced Damage. Cancer Res. 2005, 65 (21), 9799– 9806. https://doi.org/10.1158/0008-5472.CAN-05-1095.
(39) Fukuda, H.; Takamura-Enya, T.; Masuda, Y.; Nohmi, T.; Seki, C.; Kamiya, K.; Sugimura, T.; Masutani, C.; Hanaoka, F.; Nakagama, H. Translesional DNA Synthesis through a C8- Guanyl Adduct of 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b] Pyridine (PhIP) in Vitro:
95 REV1 Inserts DC Opposite the Lesion, and DNA Polymerase κ Potentially Catalyzes Extension Reaction from the 3′-DC Terminus. J. Biol. Chem. 2009, 284 (38), 25585–25597. https://doi.org/10.1074/jbc.M109.037259.
(40) Jarosz, D. F.; Godoy, V. G.; Delaney, J. C.; Essigmann, J. M.; Walker, G. C. A Single Amino Acid Governs Enhanced Activity of DinB DNA Polymerases on Damaged Templates. Nature 2006, 439 (7073), 225–228. https://doi.org/10.1038/nature04318.
(41) Hashimoto, K.; Cho, Y.; Yang, I. Y.; Akagi, J. I.; Ohashi, E.; Tateishi, S.; De Wind, N.; Hanaoka, F.; Ohmori, H.; Moriya, M. The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]Pyrene-DG and Recruitment of Polymerase ζ by REV1 to Replication-Stalled Site. J. Biol. Chem. 2012, 287 (12), 9613– 9622. https://doi.org/10.1074/jbc.M111.331728.
(42) Sharma, S.; Helchowski, C. M.; Canman, C. E. The Roles of DNA Polymerase ζ and the Y Family DNA Polymerases in Promoting or Preventing Genome Instability. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis. March 2013, pp 97– 110. https://doi.org/10.1016/j.mrfmmm.2012.11.002.
(43) Kreuzer, K. N. DNA Damage Responses in Prokaryotes: Regulating Gene Expression, Modulating Growth Patterns, and Manipulating Replication Forks. Cold Spring Harb. Perspect. Biol. 2013, 5 (11), 1–23. https://doi.org/10.1101/cshperspect.a012674.
(44) Janion, C. Inducible SOS Response System of DNA Repair and Mutagenesis in Escherichia Coli. Int. J. Biol. Sci. 2008, 4 (6), 338–344. https://doi.org/10.7150/ijbs.4.338.
(45) Nohmi, T. Environmental Stress and Lesion-Bypass DNA Polymerases. Annu. Rev. Microbiol. 2006, 60 (1), 231–253. https://doi.org/10.1146/annurev.micro.60.080805.142238.
(46) Szombati, Z.; Baerns, S.; Marx, A.; Meier, C. Synthesis of C8-Arylamine-Modified 2′-
96 Deoxyadenosine Phosphoramidites and Their Site-Specific Incorporation into Oligonucleotides. ChemBioChem 2012, 13 (5), 700–712. https://doi.org/10.1002/cbic.201100573.
(47) Gillet, L. C. J.; Scha, O. D. Preparation of C8-Amine and Acetylamine Adducts of 2′- Deoxyguanosine Suitably Protected for DNA Synthesis. 2002. https://doi.org/10.1021/ol026474f.
(48) Stover, J. S.; Rizzo, C. J. Synthesis of Oligonucleotides Containing the N 2- Deoxyguanosine Adduct of the Dietary Carcinogen 2-Amino-3-Methylimidazo[4,5- f]Quinoline. 2007. https://doi.org/10.1021/tx7002867.
(49) Schoffers, E.; Olsen, P. D.; Means, J. C. Synthesis of C8-Adenosine Adducts of Arylamines Using Palladium Catalysis. Org. Lett. 2001, 3 (26), 4221–4223. https://doi.org/10.1021/ol016900h.
(50) Colis, L. C.; Chakraborti, D.; Hilario, P.; McCarty, C.; Basu, A. K. Synthesis of Oligonucleotides Containing 2′-Deoxyguanosine Adducts of Nitropyrenes. Nucleosides, Nucleotides and Nucleic Acids 2009, 28 (2), 67–77. https://doi.org/10.1080/15257770902736426.
(51) Paul, F.; Patt, J.; Hartwig, J. F. Palladium-Catalyzed Formation of Carbon-Nitrogen Bonds. Reaction Intermediates and Catalyst Improvements in the Hetero Cross-Coupling of Aryl Halides and Tin Amides. Journal of the American Chemical Society. June 1, 1994, pp 5969– 5970. https://doi.org/10.1021/ja00092a058.
(52) Guram, A. S.; Buchwald, S. L. Palladium-Catalyzed Aromatic Animations with in Situ Generated Aminostannanes. J. Am. Chem. Soc. 1994, 116 (17), 7901–7902. https://doi.org/10.1021/ja00096a059.
(53) Heravi, M. M.; Kheilkordi, Z.; Zadsirjan, V.; Heydari, M.; Malmir, M. Buchwald-Hartwig
97 Reaction: An Overview. Journal of Organometallic Chemistry. Elsevier B.V. April 15, 2018, pp 17–104. https://doi.org/10.1016/j.jorganchem.2018.02.023.
(54) Sunesson, Y.; Limé, E.; Nilsson Lill, S. O.; Meadows, R. E.; Norrby, P. O. Role of the Base in Buchwald-Hartwig Amination. J. Org. Chem. 2014, 79 (24), 11961–11969. https://doi.org/10.1021/jo501817m.
(55) Singh, U. K.; Strieter, E. R.; Blackmond, D. G.; Buchwald, S. L. Mechanistic Insights into the Pd(BINAP)-Catalyzed Amination of Aryl Bromides: Kinetic Studies under Synthetically Relevant Conditions. J. Am. Chem. Soc. 2002, 124 (47), 14104–14114. https://doi.org/10.1021/ja026885r.
(56) Shekhar, S.; Ryberg, P.; Hartwig, J. F.; Mathew, J. S.; Blackmond, D. G.; Strieter, E. R.; Buchwald, S. L. Reevaluation of the Mechanism of the Amination of Aryl Halides Catalyzed by BINAP-Ligated Palladium Complexes. J. Am. Chem. Soc. 2006, 128 (11), 3584–3591. https://doi.org/10.1021/ja045533c.
(57) Oligo synthesis: Custom DNA oligonucleotides https://www.atdbio.com/content/6/Oligo- synthesis-Custom-DNA-oligonucleotides (accessed Nov 12, 2019).
(58) Biosystems, A. Evaluating and Isolating Synthetic Oligonucleotides. 1992, 10–11.
(59) Oligonucleotide Synthesis - an overview | ScienceDirect Topics https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular- biology/oligonucleotide-synthesis (accessed Nov 12, 2019).
(60) Dahl, B. H.; Nielsen, J.; Dahl, O. Mechanistic Studies on the Phosphoramidite Coupling Reaction in Oligonucleotide Synthesis. I. Evidence for Nudeophilic Catalysis by Tetrazole and Rate Variations with the Phosphorus Substituents. Nucleic Acids Res. 1987, 15 (4), 1729–1743. https://doi.org/10.1093/nar/15.4.1729.
98 (61) Santosh Laxmi, Y. R.; Suzuki, N.; Dasaradhi, L.; Johnson, F.; Shibutani, S. Preparation of Oligodeoxynucleotides Containing a Diastereoisomer of α-( N 2 -2‘- Deoxyguanosinyl)Tamoxifen by Phosphoramidite Chemical Synthesis. Chem. Res. Toxicol. 2002, 15 (2), 218–225. https://doi.org/10.1021/tx0101494.
(62) Attaluri, S.; Bonala, R. R.; Yang, I. Y.; Lukin, M. A.; Wen, Y.; Grollman, A. P.; Moriya, M.; Iden, C. R.; Johnson, F. DNA Adducts of Aristolochic Acid II: Total Synthesis and Site-Specific Mutagenesis Studies in Mammalian Cells. Nucleic Acids Res. 2009, 38 (1), 339–352. https://doi.org/10.1093/nar/gkp815.
(63) Bonala, R. R.; Torres, M. C.; Attaluri, S.; Iden, C. R.; Johnson, F. Incorporation of N 2 - Deoxyguanosine Metabolic Adducts of 2-Aminonaphthalene and 2-Aminofluorene into Oligomeric DNA. Chem. Res. Toxicol. 2005, 18 (3), 457–465. https://doi.org/10.1021/tx0497907.
(64) Yagi, H.; Frank, H.; Seidel, A.; Jerina, D. M. Revised Assignment of Absolute Configuration of the Cis-and Trans-N 6-Deoxyadenosine Adducts at C 14 of (()-11,12r- Dihydroxy-13r,14r-Epoxy-11,12,13,14-Tetrahydrodibenzo[a,l]Pyrene by Stereoselective Synthesis. https://doi.org/10.1021/tx800268f.
(65) Lakshman, M. K.; Keeler, J. C.; Hilmer, J. H.; Martin, J. Q. Palladium-Catalyzed C-N Bond Formation: Facile and General Synthesis of N 6-Aryl 2′-Deoxyadenosine Analogues. 1999. https://doi.org/10.1021/ja9908671.
(66) Sproviero, M.; Verwey, A. M. R.; Rankin, K. M.; Witham, A. A.; Soldatov, D. V.; Manderville, R. A.; Fekry, M. I.; Sturla, S. J.; Sharma, P.; Wetmore, S. D. Structural and Biochemical Impact of C8-Aryl-Guanine Adducts within the NarI Recognition DNA Sequence: Influence of Aryl Ring Size on Targeted and Semi-Targeted Mutagenicity. Nucleic Acids Res. 2014, 42 (21), 13405–13421. https://doi.org/10.1093/nar/gku1093.
(67) Miyahara, T.; Nakatsuji, H.; Sugiyama, H. Helical Structure and Circular Dichroism Spectra
99 of DNA: A Theoretical Study. J. Phys. Chem. A 2013, 117 (1), 42–55. https://doi.org/10.1021/jp3085556.
(68) Millen, A. L.; Sharma, P.; Wetmore, S. D. C8-Linked Bulky Guanosine DNA Adducts: Experimental and Computational Insights into Adduct Conformational Preferences and Resulting Mutagenicity. Future Med. Chem. 2012, 4 (15), 1981–2007. https://doi.org/10.4155/fmc.12.138.
(69) Berger, F. D.; Sturla, S. J.; Kung, R. W.; Montina, T.; Wetmore, S. D.; Manderville, R. A. Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl- Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation. Chem. Res. Toxicol. 2018, 31 (1), 37–47. https://doi.org/10.1021/acs.chemrestox.7b00266.
(70) Nolan, S. J.; Vyas, R. R.; Hingerty, B. E.; Euis, S.; Broyde, S.; Shapiro, R.; Basu, A. K. Solution Properties and Computational Analysis of an Oligodeoxynucleotide Containing IV-(Deoxyguanosin-8-Yl)-l-Aminopyrene; 1996; Vol. 17.
(71) Tolentino, J. H.; Burke, T. J.; Mukhopadhyay, S.; Glenn Mcgregor, W.; Basu, A. K. Inhibition of DNA Replication Fork Progression and Mutagenic Potential of 1, N 6- Ethenoadenine and 8-Oxoguanine in Human Cell Extracts. Nucleic Acids Res. 2008, 36 (4), 1300–1308. https://doi.org/10.1093/nar/gkm1157.
(72) Venkatarangan, L.; Sivaprasad, A.; Johnson, F.; Basu, A. K. Site-Specifically Located 8- Amino-2′-Deoxyguanosine: Thermodynamic Stability and Mutagenic Properties in Escherichia Coli; 2001; Vol. 29.
(73) Jain, V.; Hilton, B.; Lin, B.; Patnaik, S.; Liang, F.; Darian, E.; Zou, Y.; MacKerell, A. D.; Cho, B. P. Unusual Sequence Effects on Nucleotide Excision Repair of Arylamine Lesions: DNA Bending/Distortion as a Primary Recognition Factor. Nucleic Acids Res. 2013, 41 (2), 869–880. https://doi.org/10.1093/nar/gks1077.
100 (74) Mu, H.; Kropachev, K.; Wang, L.; Zhang, L.; Kolbanovskiy, A.; Kolbanovskiy, M.; Geacintov, N. E.; Broyde, S. Nucleotide Excision Repair of 2-Acetylaminofluorene-and 2- Aminofluorene-(C8)-Guanine Adducts: Molecular Dynamics Simulations Elucidate How Lesion Structure and Base Sequence Context Impact Repair Efficiencies. Nucleic Acids Res. 2012, 40 (19), 9675–9690. https://doi.org/10.1093/nar/gks788.
(75) Kung, R. W.; Sharma, P.; Wetmore, S. D. Effect of Size and Shape of Nitrogen-Containing Aromatics on Conformational Preferences of DNA Containing Damaged Guanine. J. Chem. Inf. Model. 2018, 58 (7), 1415–1425. https://doi.org/10.1021/acs.jcim.8b00238.
(76) Kathuria, P.; Sharma, P.; Wetmore, S. D. Adenine versus Guanine DNA Adducts of Aristolochic Acids: Role of the Carcinogen-Purine Linkage in the Differential Global Genomic Repair Propensity. Nucleic Acids Res. 2015, 43 (15), 7388–7397. https://doi.org/10.1093/nar/gkv701.
(77) Huchting, J.; Winkler, M.; Nasser, H.; Meier, C. Synthesis of T-705-Ribonucleoside and T- 705-Ribonucleotide and Studies of Chemical Stability. ChemMedChem 2017, 12 (9), 652– 659. https://doi.org/10.1002/cmdc.201700116.
(78) Mao, B.; Vyas, R. R.; Hingerty, B. E.; Broyde, S.; Basu, A. K.; Patel, D. J. Articles Solution Conformation of the N-(Deoxyguanosin-8-Yl)-1-Aminopyrene ([AP]DG) Adduct Opposite DC in a DNA Duplex †; 1996.
(79) Patel, D. J.; Mao, B.; Gu, Z.; Hingerty, B. E.; Gorin, A.; Basu, A. K.; Broyde, S. Invited Review Nuclear Magnetic Resonance Solution Structures of Covalent Aromatic Amine- DNA Adducts and Their Mutagenic Relevance; 1998.
(80) Sharma, P.; Manderville, R. A.; Wetmore, S. D. Modeling the Conformational Preference of the Carbon-Bonded Covalent Adduct Formed upon Exposure of 2′-Deoxyguanosine to Ochratoxin A. Chem. Res. Toxicol. 2013, 26 (5), 803–816. https://doi.org/10.1021/tx4000864.
101 (81) Höfler, K.; Sarac, I.; Meier, C. Synthesis of C8-N-Arylamine-Modified 2′- Deoxyguanosine-5′-Triphosphates and Their Effects on Primer Extension by DNA Polymerases. ChemBioChem 2015, 16 (14), 2046–2053. https://doi.org/10.1002/cbic.201500293.
(82) Hou, H.; Zhao, Y.; Li, C.; Wang, M.; Xu, X.; Jin, Y. Single-Cell PH Imaging and Detection for PH Profiling and Label-Free Rapid Identification of Cancer-Cells. Sci. Rep. 2017, 7 (1), 1–8. https://doi.org/10.1038/s41598-017-01956-1.
(83) Sun, Y. W.; Guttenplan, J. B.; Khmelnitsky, M.; Krzeminski, J.; Boyiri, T.; Amin, S.; El- Bayoumy, K. Stereoselective Metabolism of the Environmental Mammary Carcinogen 6- Nitrochrysene to Trans-1,2-Dihydroxy-1,2-Dihydro-6-Nitrochrysene by Aroclor 1254- Treated Rat Liver Microsomes and Their Comparative Mutation Profiles in a LacI Mammary Epithelial Cell Line. Chem. Res. Toxicol. 2009, 22 (12), 1992–1997. https://doi.org/10.1021/tx9002897.
(84) Moriya, M. Single-Stranded Shuttle Phagemid for Mutagenesis Studies in Mammalian Cells: 8-Oxoguanine in DNA Induces Targeted G·C → T·A Transversions in Simian Kidney Cells. Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (3), 1122–1126. https://doi.org/10.1073/pnas.90.3.1122.
(85) Pande, P.; Haraguchi, K.; Jiang, Y. L.; Greenberg, M. M.; Basu, A. K. Unlike Catalyzing Error-Free Bypass of 8-OxodGuo, DNA Polymerase λ Is Responsible for a Significant Part of Fapy·dG-Induced G → T Mutations in Human Cells. Biochemistry 2015, 54 (10), 1859– 1862. https://doi.org/10.1021/acs.biochem.5b00119.
(86) Bose, A.; Pande, P.; Jasti, V. P.; Millsap, A. D.; Hawkins, E. K.; Rizzo, C. J.; Basu, A. K. DNA Polymerases κ and ζ Cooperatively Perform Mutagenic Translesion Synthesis of the C8-2’-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells. Nucleic Acids Res. 2015, 43 (17), 8340–8351. https://doi.org/10.1093/nar/gkv750.
102 (87) Abul Kalam, M.; Haraguchi, K.; Chandani, S.; Loechler, E. L.; Moriya, M.; Greenberg, M. M.; Basu, A. K. Genetic Effects of Oxidative DNA Damages: Comparative Mutagenesis of the Imidazole Ring-Opened Formamidopyrimidines (Fapy Lesions) and 8-Oxo-Purines in Simian Kidney Cells. Nucleic Acids Res. 2006, 34 (8), 2305–2315. https://doi.org/10.1093/nar/gkl099.
(88) Weerasooriya, S.; Jasti, V. P.; Basu, A. K. Replicative Bypass of Abasic Site in Escherichia Coli and Human Cells: Similarities and Differences. PLoS One 2014, 9 (9). https://doi.org/10.1371/journal.pone.0107915.
(89) Du, H.; Wang, P.; Li, L.; Wang, Y. Repair and Translesion Synthesis of O 6 -Alkylguanine DNA Lesions in Human Cells . J. Biol. Chem. 2019, 294 (29), 11144–11153. https://doi.org/10.1074/jbc.ra119.009054.
(90) Wu, J.; Li, L.; Wang, P.; You, C.; Williams, N. L.; Wang, Y. Translesion Synthesis of O4- Alkylthymidine Lesions in Human Cells. Nucleic Acids Res. 2016, 44 (19), 9256–9265. https://doi.org/10.1093/nar/gkw662.
(91) Delaney, J. C.; Essigmann, J. M. Assays for Determining Lesion Bypass Efficiency and Mutagenicity of Site-Specific DNA Lesions In Vivo. Methods in Enzymology. 2006, pp 1– 15. https://doi.org/10.1016/S0076-6879(06)08001-3.
(92) Weerasooriya, S.; Jasti, V. P.; Bose, A.; Spratt, T. E.; Basu, A. K. Roles of Translesion Synthesis DNA Polymerases in the Potent Mutagenicity of Tobacco-Specific Nitrosamine- Derived O2-Alkylthymidines in Human Cells. DNA Repair (Amst). 2015, 35, 63–70. https://doi.org/10.1016/j.dnarep.2015.09.023.
(93) You, C.; Swanson, A. L.; Dai, X.; Yuan, B.; Wang, J.; Wang, Y. Translesion Synthesis of 8,5’-Cyclopurine-2’-Deoxynucleosides by DNA Polymerasesη,ℓ, Andζ. J. Biol. Chem. 2013, 288 (40), 28548–28556. https://doi.org/10.1074/jbc.M113.480459.
103 (94) Sakuma, T.; Nishikawa, A.; Kume, S.; Chayama, K.; Yamamoto, T. Multiplex Genome Engineering in Human Cells Using All-in-One CRISPR/Cas9 Vector System. Sci. Rep. 2014, 4. https://doi.org/10.1038/srep05400.
(95) Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; DiCarlo, J. E.; Norville, J. E.; Church, G. M. RNA-Guided Human Genome Engineering via Cas9. Science (80-. ). 2013, 339 (6121), 823–826. https://doi.org/10.1126/science.1232033.
(96) Beckman, K. B.; Ames, B. N. Oxidative Decay of DNA. Journal of Biological Chemistry. August 8, 1997, pp 19633–19636. https://doi.org/10.1074/jbc.272.32.19633.
(97) Cooke, M. S.; Evans, M. D.; Dizdaroglu, M.; Lunec, J. Oxidative DNA Damage: Mechanisms, Mutation, and Disease. FASEB Journal. July 2003, pp 1195–1214. https://doi.org/10.1096/fj.02-0752rev.
(98) Greenberg, M. M. The Formamidopyrimidines: Purine Lesions Formed in Competition With 8-Oxopurines From Oxidative Stress. Acc. Chem. Res. 2012, 45 (4), 588–597. https://doi.org/10.1021/ar2002182.
(99) Téoule, R. Radiation-Induced DNA Damage and Its Repair. International Journal of Radiation Biology. Informa Healthcare 1987, pp 573–589. https://doi.org/10.1080/09553008414552111.
(100) Biswas, P. K.; Chakraborty, S. Targeted DNA Oxidation and Trajectory of Radical DNA Using DFT Based QM/MM Dynamics. Nucleic Acids Res. 2019, 47 (6), 2757–2765. https://doi.org/10.1093/nar/gkz089.
(101) Breen, A. P.; Murphy, J. A. Reactions of Oxyl Radicals with DNA. Free Radical Biology and Medicine. 1995, pp 1033–1077. https://doi.org/10.1016/0891-5849(94)00209-3.
(102) Candeias, L. P.; Steenken, S. Reaction of HO. with Guanine Derivatives in Aqueous
104 Solution: Formation of Two Different Redox-Active OH-Adduct Radicals and Their Unimolecular Transformation Reactions. Properties of G(-H). Chem. - A Eur. J. 2000, 6 (3), 475–484.https://doi.org/10.1002/(SICI)1521-3765(20000204)6:3<475::AID- CHEM475>3.0.CO;2-E.
(103) Lin Jiang, Y.; Wiederholt, C. J.; Patro, J. N.; Haraguchi, K.; Greenberg, M. M. Synthesis of Oligonucleotides Containing Fapy‚dG (N 6-(2-Deoxy-r,-D-Erythropentofuranosyl)-2,6- Diamino-4-Hydroxy-5-Formamidopyrimidine) Using a 5′-Dimethoxytrityl Dinucleotide Phosphoramidite. Angew. Chem., Int. Ed 2000, 408 (1), 141–149. https://doi.org/10.1021/jo048253o.
(104) Haraguchi, K.; Delaney, M. O.; Wiederholt, C. J.; Sambandam, A.; Hantosi, Z.; Greenberg, M. M. Synthesis and Characterization of Oligodeoxynucleotides Containing Formamidopyrimidine Lesions and Nonhydrolyzable Analogues. J. Am. Chem. Soc. 2002, 124 (13), 3263–3269. https://doi.org/10.1021/ja012135q.
(105) Haraguchi, K.; Greenberg, M. M. In ComprehensiVe Natural Products Chemistry. J. Chem. Soc., Perkin Trans. 1 1999, 111 (9), 8636–8637. https://doi.org/10.1021/ja0160952.
(106) Hollstein, M.; Rice, K.; Greenblatt, M. S.; Soussi, T.; Fuchs, R.; Sørlie, T.; Hovig, E.; Smith-Sørensen, B.; Montesano, R.; Harris, C. C. Database of P53 Gene Somatic Mutations in Human Tumors and Cell Lines. Nucleic Acids Res. 1994, 22 (17), 3551–3555.
(107) Hollstein, M.; Shomer, B.; Greenblatt, M.; Soussi, T.; Hovig, E.; Montesano, R.; Harris, C. C. Somatic Point Mutations in the P53 Gene of Human Tumors and Cell Lines: Updated Compilation; 1996; Vol. 24.
(108) Kino, K.; Saito, I.; Sugiyama, H. Product Analysis of GG-Specific Photooxidation of DNA via Electron Transfer: 2-Aminoimidazolone as a Major Guanine Oxidation Product; Kluwer Academic Publishers, 1990; Vol. 1.
105 (109) Hussain, S. P.; Harris, C. C. Molecular Epidemiology of Human Cancer: Contribution of Mutation Spectra Studies of Tumor Suppressor Genes. Cancer Research. September 15, 1998, pp 4023–4037.
(110) Hussain, S. P.; Raja, K.; Amstad, P. A.; Sawyer, M.; Trudel, L. J.; Wogan, G. N.; Hofseth, L. J.; Shields, P. G.; Billiar, T. R.; Trautwein, C.; et al. Increased P53 Mutation Load in Nontumorous Human Liver of Wilson Disease and Hemochromatosis: Oxyradical Overload Diseases. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (23), 12770–12775. https://doi.org/10.1073/pnas.220416097.
106
Appendix A: Chapter 2 Supporting Information – Spectral Data
107
Figure S1. 1H NMR spectrum of 8-bromo-2’-deoxyadenosine
108
Figure S2. 13C NMR spectrum of 8-bromo-2’-deoxyadenosine
109 [M − dR + H]+
NH2
N N Br N N HO O
OH
+ + m/z calculated for [M+H] C10H12BrN5O3 : 330.14 m/z observed : 330.0188
Figure S3. MS spectrum of 8-bromo-2’-deoxyadenosine
110
4
Figure S4.1H NMR spectrum of 8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (4)
111 NH2
N N Br N N TBDMS-O O
O-TBDMS
+ m/z calculated for C22H40BrN5O3Si2 : 558.67 m/z observed: 558. 1917
Figure S5. MS spectrum of 8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (4)
112
5
6
Figure S6. 1H NMR spectrum of 8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (5)
113
5
6
Figure S7. 13C NMR of 8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (5)
114 +TOF MS: 0.032 to 0.082 min from Sample 1 (TuneSampleID) of 011816-Brent-Protected dG.wiff Max. 3200.8 counts. a=3.60144582785556810e-004, t0=6.37966570470817090e+001 (Turbo Spray)
862.3206 3200
3000
2800 860.3245 Protected dA, [MH]+, calc. 860.3238 2600 5
2400
2200
2000
1800 863.3247 861.3281
1600
Intensity, counts Intensity, 1400
1200
1000
800
600 864.3257 400
200 816.3815 782.4207 818.3811 865.3300 0 770 775 780 785 790 795 800 805 810 815 820 825 830 835 840 845 850855 860 865 870 875 880 885 m/z, Da
Figure S8. MS spectrum of 8-bromo-3’, 5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (5)
115
6b
Figure S9. 1H NMR spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (6b)
116
6b
Figure S10. 1H NMR spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (6b)
117
6b
Figure S11. MS spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-N6-dimethoxytrityl-2’-deoxyadenosine (6b)
118
6a
Figure S12. 1H NMR spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (6a)
119
6a
Figure S13. 13C NMR spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (6a)
120 +TOF MS: 0.049 to 0.682 min from Sample 1 (TuneSampleID) of 060815-BVP-DMT-ClE.wiff Max. 1017.0 counts. a=3.60041086922528760e-004, t0=6.27962639916586340e+001 (Turbo Spray)
721.3729 1000
950
900
850
800
750
700
650
600 BVP-DMT-ClE, [M+H]+, calc. 721.3718
550 6a
500 722.3746
450 Intensity, counts Intensity,
400
350
300
250
200 723.3747 150
100
50 724.3758
0 580 600 620 640 660 680 700 720 740 760 780 800 820 840 m/z, Da Figure S14. MS spectrum of 8N-(6-aminochrysene)-3’,5’-O-bis(tert-butyldimethylsilyl)-2’-deoxyadenosine (6a)
121
3
Figure S15. 1H NMR spectrum of 8N-(6-aminochrysene)-2’-deoxyadenosine (3)
122
3
Figure S16. 13C NMR spectrum of 8N-(6-aminochrysene)-2’-deoxyadenosine (3)
123
NH2
N N HN N N HO O
OH
Figure S17. 1H-1H ROESY NMR spectrum of 8N-(6-aminochrysene)-2’-deoxyadenosine (3)
124
Figure S18. MS spectrum of 8N-(6-aminochrysene)-2’-deoxyadenosine (3)
125
Figure S19. 1H spectrum of N6-Benzoyl-8N-(aminochrysene)-2'-deoxyadenosine (7)
126
Figure S20. 13C spectrum of N6-Benzoyl-8N-(aminochrysene)-2'-deoxyadenosine (7)
127 +TOF MS: 3.348 to 3.581 min from Sample 1 (TuneSampleID) of 061616-Brent-Ben-DPR-OH.wiff Max. 1.4e4 counts. a=3.60095417166520350e-004, t0=6.43268010587999020e+001 (Turbo Spray) 597.2234 240
230
220 210 Ben-DPR-OH, [MH]+, calc. 597.2250 200 [M+Na]+ 7 190
180 619.2051 170
160
150
140
130
120
110 Intensity, counts Intensity, 100
90 598.2288 80
70
60 620.2097 50
40
30
20
10
0 530 540 550 560 570 580 590 600 610 620 630 640 650 660 670 680 690 700 710 720 730 m/z, Da Figure S21. MS spectrum of N6-Benzoyl-8N-(aminochrysene)-2'-deoxyadenosine (7)
128 +TOF MS: 1.332 to 1.615 min from Sample 1 (TuneSampleID) of 080216-Brent-5-DMTr.wiff Max. 6184.9 counts. a=3.60083540110247080e-004, t0=6.44753197454556360e+001 (Turbo Spray)
899.3526 320
300
280
260 O
240 HN Ph 5'DMTr, [MH]+, calc. 899.3552 220 N N HN 200 N N DMTrO 180 O 900.3574
160 OH
Intensity, counts Intensity, 140
120 + m/z caluculated for C56H46N6O6 : 899.35 observed : 899.3526 100
80
60
901.3615 40
20
0 810 820 830 840 850 860 870 880 890 900 910 920 930 940 950 960 970 980 990 1000 m/z, Da
Figure S22. MS spectrum of N6-Benzoyl-8N-(6-aminochrysene)-5'-O-dimethoxytrityl-2'-deoxyadenosine
129
Figure S23. 31P spectrum of N6-Benzoyl-8N-(6-aminochrysene)-5'-O-dimethoxytrityl-2'-deoxyadenosine-3'-O- (cyanoethyl-N,N'-diisopropyl-phosphoramidite (8)
130
+TOF MS: 2.235 to 2.385 min from Sample 1 (TuneSampleID) of 101116-Brent-Phosphoramidile.wiff Max. 1.3e4 counts. a=3.60485127759944080e-004, t0=6.35676267610148930e+001 (Turbo Spray)
1099.4607 210 O 200
190 HN Ph
180 N N HN 170 Phosphoramidile, [MH]+, calc. 1099.4636 N N 160 DMTr O 1100.4653 150 O 140 130 CN O P 120 N 110
100 Intensity, counts Intensity, 90 + m/z calculated for C65H63N8O7P : 1099.46 80 observed : 1099.4606 70
60 + 1101.4705 [M+Na] 50
40 1121.4446
30 1122.4488 1115.4575
20 1116.4604 1102.4718 10
0 1060 1065 1070 1075 1080 1085 1090 1095 1100 1105 1110 1115 112011251130113511401145 m/z, Da Figure S24. MS spectrum of N6-Benzoyl-8N-(6-aminochrysene)-5'-O-dimethoxytrityl-2'-deoxyadenosine-3'-O- (cyanoethyl-N,N'-diisopropyl-phosphoramidite (8)
131 jlb20170614_BPowell_12mer2_1uM_1 #502-524 RT: 0.60-0.62 AV: 23 NL: 1.97E6 jlb20170614_BPowell_12mer2_1uM_1 #502-524 RT: 0.60-0.62 AV: 23T: FTMSNL: - p 1.97E6ESI Full ms [300.0000-4000.0000] T: FTMS - p ESI5' Full-GTGCA*TGTTTGT ms [300.0000-4000.0000] -3' 1306.5697 z=3 1306.5697 100 3- 1306.9022 (M-3H) : 1306.2352 m/z yields z=3 95 z=3 100 1307.5683 90 an overall MW of 3921.7274 Da 95 z=3 85 80 90 75 85 70 65 1306.2352 1307.2349 z=3 z=3 80 60 75 55 50 70 45 40 RelativeAbundance 1307.5683 65 35 z=3 60 30 25 55 20 1307.9015 z=3 50 15 10 1308.2396 45 5 1305.9397 z=3 1308.5650 z=? z=3 0 40 1319.2190 1306.0 1306.5 1307.0 1307.5 1308.0 1308.5 1309.0 RelativeAbundance z=3 m/z 35 1313.8952 z=3 1326.5453 30 z=3 25 1315.2336 1327.8817 z=3 z=3 20 1332.2021 1339.5393 1321.2226 15 z=3 z=3 z=3 1346.5299 1337.5278 1340.8700 1355.9363 10 z=3 1352.5212 z=? 1312.2450 z=3 z=3 z=3 1359.8540 1364.5171 5 z=3 z=3 z=3 0 1305 1310 1315 1320 1325 1330 1335 1340 1345 1350 1355 1360 1365 m/z
Figure S25. ESI-MS (negative mode) spectrum of N-(dA-8-yl)-6-AC modified 12-mer oligodeoxynucleotide
132 (M-4H)4-: 1195.2252 m/z yields an jlb20170614_BPowell_15mer_1uM_15'-GCCCTCAA*CAAGATG #578-602 RT: 0.68-0.71 AV: 25 NL: -1.09E63' T: FTMS - p ESI Full ms [333.4000-5000.0000] jlb20170614_BPowell_15mer_1uM_1 #578-602 RT: 0.68-0.71 AV: 25 NL: 1.09E6 overall MW of 4784.9298 Da 1195.7207 T: FTMS - p ESI Full ms [333.4000-5000.0000] 1195.7207 z=4 z=4 100 100 95 1195.4710 95 z=4 90
90 85 1195.9702 z=4 85 1196.4678 80 z=4 75 80 70 75 65 60 70 55 1196.2193 50 z=4 65 1195.2252 45 z=4 60 40 RelativeAbundance 55 35 1196.4678 30 50 z=4 25 45 20 15 1196.7221 40 z=4 RelativeAbundance 10 1196.9724 35 5 z=4 1197.2194 z=4 0 30 1194.8 1195.0 1195.2 1195.4 1195.6 1195.8 1196.0 1196.2 1196.4 1196.6 1196.8 1197.0 1197.2 1197.4 25 1205.2092 m/z z=4 20 1201.2163 1209.9550 15 z=4 z=4 1206.4617 1214.6967 1224.1840 10 1199.9769 1219.9425 1233.6721 z=4 z=4 z=4 z=4 1229.6795 z=4 1239.4206 5 z=4 z=4 z=4 0 1195 1200 1205 1210 1215 1220 1225 1230 1235 1240 m/z
Figure S26. ESI-MS (negative mode) spectrum of N-(dA-8-yl)-6-AC modified 15-mer oligodeoxynucleotide
133 CT Adduct CT Adduct
mer mer mer mer 12 12 15 15 (-)
Lane 1 5'-GTGCATGTTTGT-3’
Lane 2 5'-GTGCA*TGTTTGT-3’
Lane 3 5'-GCCCTCAACAAGATG-3’
Lane 4 5'-GCCCTCAA*CAAGATG-3’
(+)
1 2 3 4
Figure S27. 20% PAGE Gel of Pure N-(dA-8-yl)-6-AC modified 12-mer and 15-mer oligodeoxynucleotides
134
Appendix B: Chapter 4 Supporting Information
135
Table S1. Mutational frequency in wild type (-) SOS and (+) SOS E. coli (AB1157) cells
5’-G1C1C2 C3T1 C4A1 A*2C3A3A4G2ATG3-3’
Replication in E. coli Strain AB1157 Total Total % Exp # colonies no. of MF (%) Survival Targeted Mutations Semi-Targeted Mutations screened mutants [no. of mutants] (%)
A*→T A*→G A*→C A*del (%) (%) (%) (%)
1 (-) SOS 149 9 6.0 65** 0 4 0 0 5’-A1A*2C →TGC [4] (2.7) -- (2.68) -- -- 5’-G1C1C2→ GGC [1] (0.7) 5’-A3A4G2→ _AG [4] (2.7)
2 (-) SOS 98 3 3.0 62* 0 2 0 0 5’-A1A*2C →TGC [2] (2.0) -- (2.04) -- -- A3A4G2→ _AG [1] (1.0)
3 (-) SOS 265 15 5.6 58*** 2 6 0 0 A1A*2C →TGC [2] (0.8) (0.75) (2.26) -- -- 5’-G1C1C2→ GGC [1] (0.3) A*2C3A3A4G2ATG del. [3] (1.1) 5’-C4A1 A*2 deletion [1] (0.8) C3 del [1] (0.3) 5’-A*2C3A3A4G2ATG [1] (0.3)
136 4 (-) SOS 158 8 5.0 51* 1 4 0 1 A1A*2C →TGC [1] (0.6) (0.6) (2.53) -- (0.6) A1 or A*2 del [1] (0.6)
(-) SOS 670 35 5.2% 59 3 16 0 1 16 TOTALS (0.45%) (2.4%) -- -- (2.4%)
1 183 25 13.6 89*** 3 9 7 0 G1→ C [1] (0.54) (+) SOS (1.64) (4.92) (3.82) -- G3→ A [1] (0.54) G3 del [1] (0.54) C3T1 C4 → TT1A [1] (0.54) 5’-C3T1 C4A1 del [2] (1.09)
2 127 13 10.23 85*** 2 7 1 0 5’-C3A3A4 del [1] (0.79) (+) SOS (1.57) (5.51) (0.78) -- T1 del [1] (0.79)
C1→ A [1] (0.79) (+) SOS 310 38 12.2% 87 5 16 8 0 10 TOTALS (1.61) (5.16) (2.58) -- (3.2%)
*3 Plates Average; **3 – 6 Plates Average; ***6 – 12 Plates Average
137
Table S2. TLS efficiencies and mutational frequencies in HEK WT and KO cells
5’-G1C1C2C3T1C4A1 A*2C5A3A4G2A5TG-3’ Replication in HEK 293T WT cells Exp # # of Mutants MF (%) % Targeted Mutations Semi-Targeted Mutations Colonies Survival [# of mutants] (%) Screened A*→T A*→G A*→C A* del 1 88 15 17.0 --- 6 5 1 0 C3 →T [1] (1.1) HEK293T C1→ G [1] (1.1) T2 del [1] (1.1)
2 289 36 12.4 87* 23 7 2 0 C3 →T [1] (1.1) HEK293T C1→ G [1] (1.1) T2 del [1] (1.1)
3 512 59 11.5 82** 38 14 2 0 A1 del [2] (0.4) HEK293T A1→ T [1] (0.2) C5 →T [2] (0.4)
4 257 25 9.7 79*** 13 8 1 0 A1 →T [2] (0.8) HEK293T A1 del [1] (0.4)
WT 80 34 6 0 14 Totals 1146 135 11.8% 83% [7.0%] [2.96%] [0.52%] [1.22%]
Replication in HEK 293T Pol K KO cells
138 1 376 15 4.0 --- 4 5 2 0 A1→T [1] (0.6) Pol K KO C5 del [1] (0.6) A3A4G2 [2] (1.7)
2 489 13 2.7 62*** 3 8 0 0 T1C4 del [1] (0.2) C del [1] (0.2) Pol K 5 KO
3 324 10 3.0 58** 1 6 1 0 A3A4 del [1] (0.3) A → T [1] (0.3) Pol K 1 KO Pol K- 8 19 3 0 8 Totals 1189 38 3.2% 60% [0.7%] [1.6%] [0.25%] [0.7%]
Replication in HEK 293T Pol Z KO Cells
1 266 10 3.8 70** 7 1 0 1 C5 → T [1] (0.4) Pol Z KO
2 197 8 4.0 73* 5 2 0 0 A1A2* del [1] (0.5) Pol Z KO Pol Z- 12 3 0 1 2 Totals 463 18 3.88% 71.5% [2.6%] [0.64%] [0.2%] 0.4%
Replication in HEK 293T Pol I KO Cells
1 218 24 11.2 77* 11 8 2 0 A1→ T [2] (0.9) C A del [1] (0.5) Pol I 4 1
139 KO
2 189 18 9.7 81* 9 6 0 0 C4A1A*2 del [1] (0.5) Pol I T1C4 del [1] (0.5) KO A1→ T [1] (0.5)
3 102 10 9.8 81* 4 3 1 0 A3A4 del [2] (0.3)
Pol I KO Pol I- 24 17 3 8 Totals 509 52 10.2% 80% [4.7%] [3.3%] [0.6%] [1.6%]
Replication in HEK 293T Pol H KO Cells
1 212 38 17.9 47*** 21 10 0 0 C2C3T1C4A1 A*2del [2] (0.9) C → G [1] (0.5) Pol H 1 A1→ T [3] (1.4) KO C5 del [1] (0.5)
2 165 29 18.3 42** 15 8 0 0 C5 → T [2] (1.2) Pol H A1→ T [1] (0.6) KO C5 del [1] (0.6)
Pol H- 36 18 0 0 13 Totals 377 67 17.7% 45% [9.5%] [4.8%] [3.4%]
Replication in HEK 293T Pol H/Z DKO Cells
140 1 278 13 4.8 32*** 10 1 0 0 A1→ T [1] (0.4) C del [1] (0.4) Pol H/Z 5 KO
2 320 14 4.4 34*** 9 2 0 0 C5 → T [2] (0.6) Pol H/Z A1 del [1] (0.3) KO
3 450 18 4.0 36*** 14 2 0 0 C5 → T [2] (0.4) Pol H/Z
Pol H/Z- 1048 46 4.4% 35% 33 5 1 0 7 Totals [3.14%] [0.48%] [0.09%] [0.66%]
Replication in HEK 293T Pol H/K DKO Cells
1 240 11 4.7 27** 8 1 0 0 C4A1 del [2] (0.8) Pol H/K
2 15 5.3 25** 12 0 0 0 C5 → T [1] (0.3) Pol H/K 290 A1→ T [2] (0.3)
3 10 5.0 25** 9 1 0 0 Pol H/K 198
Pol H/K- 728 36 5.0% 25.6% 29 2 0 0 5 [3.98%] [0.3%] [0.7%]
Replication in HEK 293T Pol K/Z KD Cells
141 1 670 7 1.04 55** 5 1 0 0 C5 → T [1] (0.15) Pol Z/K
2 503 5 0.99 51*** 3 1 0 0 C4 del [1] (0.2) Pol Z/K
3 278 4 1.43 56** 4 0 0 0 0 Pol Z/K
Pol Z/K 1451 16 1.1% 54 12 2 0 0 2 [0.83] [0.14] [0.14]
142
Appendix C: Chapter 5 Supporting Information
143
5’-AAC CG1*G AG3*G CCC-3’ No. of % Semi-Targeted Mutations Exp # colonies Mutants MF (%) Survival Targeted Mutations [# of mutants] screened G1- Codon 248 G*→T G*→C G*→A G* del 1 250 33 13.5 --- 19 11 2 0 C2C3C4 del [1] HEK293T
2 210 18 8.57 92** 12 4 1 0 C1→ G [1] HEK293T
3 280 36 13.0 88** 22 9 3 0 A1A2 del [1] HEK293T C1 del [1]
WT 53 24 6 0 4 Totals 740 87 12 % 90% [7.1 %] [3.2 %] [0.8 %] [0.54 %]
G3- Codon 249 G*→T G*→C G*→A G* del
1 212 13 6.1 --- 9 3 0 0 AG3*G CCC del [1] HEK293T
2 260 14 5.3 85** 11 2 0 0 C5 del [1]
HEK293T
144 3 330 19 5.9 90** 14 4 1 0 HEK293T
WT 802 45 5.7 % 88% 34 9 1 0 2 Totals [4.2 %] [1.1 %] [0.124%] [0.25%]
145 Fapy.dG PAGE Gel
1-p53 CT 5’-GTG CGT GTT TGT 2-p53 CT 5’-GTG CGT GTT TGT 3-HY2169-1 5’-AAC CXG AGG CCC 4-HY2169-2 5’-AAC CGG AXG CCC 1 2 3 4 5 5-HY2169-3 5’-GTG CXT GTT TGT
X = Fapy.dG
146