Exposure to many chemotherapeutic agents results in DNA damage that frequently occurs at the N7 position of guanine (N7-Gua). Although these adducts can serve as biomarkers for these treatments, as well as exposures to environmental carcinogens, there are no data supporting a role for N7-Gua adducts in promoting mutagenesis. However, there are compelling data that implicate the ring-opened form of alkylated N7-Gua adducts, N-alkyl-2,6-diamino-4-hydroxyformamido-pyrimidine (alkyl-Fapy) in mutagenesis and carcinogenesis. The goal of the Program Project is to determine the structural, molecular, and cellular bases for mutagenesis caused by a variety of alkyl-Fapy-Gua adducts that are produced during therapeutic treatments with temozolomide, thioTEPA and nitrogen mustard. The goals of Project 2 are to investigate the mutagenic potential of these adducts, identify the DNA polymerase(s) that generate errors as a consequence of lesion bypass, and invesfigate the repair systems that modulate mutation frequencies and spectra.
Specific Aim 1 will establish the mutagenic potential of these Fapy-dG DNA adducts by evaluating the frequency and spectra of mutations formed following replication in wild-type primate cells. It is hypothesized that both the mutation frequency and spectra will be significantly modulated by the local sequence context of the adduct, the steady-state stereochemical equilibrium ofthe adduct at the replication fork, and the size of the N-alkyl-Fapy-dG adduct. The roles of various DNA mismatch repair mechanisms in limifing mutagenesis will also be assessed. The objective of Specific Aim 2 is to identify the DNA polymerases that are responsible for the low and high fidelity bypass of the N-alkyl-Fapy adducts. It is hypothesized that pol K and possibly pol n are primarily responsible for TLS of these adducts and this will be tested using a multidisciplinary approach of shRNA-mediated depletion of individual polymerases and specific small molecule inhibitors. The objective of Specific Aim 3 is to determine the DNA repair mechanism(s) that modulate the mutagenic potential of N-alkyl-Fapy-dG adducts. It is hypothesized that base and nucleotide excision repair will modulate mutagenesis in cells that are shRNA-depleted for repair proteins.
Specific aim 4 will investigate repair and replication of DNAs containing interstrand DNA cross-links that arise from nitrogen mustards. It is hypothesized that interactions between DNA repaired translesion synthesis polymerases can leed to cellular resistance to these agents.
In order to understand the mechanisms by which cells respond to and tolerate DNA damages caused by common chemotherapeutic alkylating agents and the potential for secondary tumor formation, this study will determine the structural, molecular, and cellular bases for mutagenesis caused by the N-alkyl-dG adducts. These adducts are proposed to be a major contributor to the carcinogenic and/or mutagenic effects of exposure to many commonly used chemotherapeutic alkylating agents.
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|Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina et al. (2016) Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase Î¹. J Biol Chem 291:21063-21073|
|Egli, Martin (2016) Diffraction Techniques in Structural Biology. Curr Protoc Nucleic Acid Chem 65:7.13.1-7.13.41|
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|Patra, Amritraj; Su, Yan; Zhang, Qianqian et al. (2016) Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase Î·. J Biol Chem 291:14134-45|
|Su, Yan; Egli, Martin; Guengerich, F Peter (2016) Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase Î·. J Biol Chem 291:3747-56|
|Thiaville, Jennifer J; Kellner, Stefanie M; Yuan, Yifeng et al. (2016) Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci U S A 113:E1452-9|
|Patra, Amitraj; Zhang, Qianqian; Guengerich, F Peter et al. (2016) Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase Î·. J Biol Chem 291:24304-24313|
|O'Flaherty, Derek K; Guengerich, F Peter; Egli, Martin et al. (2015) Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase Î· opposite Intrastrand Cross-Linked DNA. Biochemistry 54:7449-56|
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