Many carcinogens become covalently attached to DNA and cause genotoxic damage due to polymerase blockage or nucleotide misincorporation. These events are controlled kinetically by the individual DNA polymerases. Continued studies are proposed on the interaction of a series of carcinogens, bound to oligonucleotides, with a set of both replicative and so-called translesion bypass polymerases, including viral (DNA polymerase T7-, HIV-1 reverse transcriptase) and bacterial (Dpo4) model polymerases as well as recombinant human polymerases (delta, eta, iota, kappa, REV1). Kinetic analyses will be done with these polymerases and the 22 DNA-carcinogen adducts available, varying in size from oxygen and methyl groups to large polycyclic hydrocarbons, identifying changes in rate-limiting events related to normal incorporation vs. misincorporation and blocking. The working hypothesis is that alternate conformations and inactive complexes are important, and some of these may also have rapid nucleotide dissociation kinetics. Another use of kinetic analysis will be to understand the chronology and coupling of the events involved in changing polymerases at DNA damage sites, i.e. between replicative and translesion polymerases. Roles of accessory proteins will be considered. Pre-steady-state kinetic spectroscopic approaches will also be used to better define events related to individual steps of polymerase catalysis, with fluorescence and circular dichroism changes being compared to measured rates of product formation. Work on the crystallography of carcinogen-adducted DNA with polymerases (HIV-1 reverse transcriptase, Dpo4) is in progress and will be expanded, with the goal of linking temporal events with specific structural changes. The general working hypothesis is that normal incorporation, misincorporation, pausing, and blockage represent a continuum of events related to fits and rates of reactions involving polymerase-DNA-dNTP ternary complexes, and that these can be understood in quantitative terms (rate constants and structures). The overall goal is understanding molecular mechanisms of mutagenesis and relevance to chemical carcinogenesis. ? ?

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Cancer Etiology Study Section (CE)
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Shaughnessy, Daniel
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Vanderbilt University Medical Center
Schools of Medicine
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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 ?. J Biol Chem 291:21063-21073
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 ?. J Biol Chem 291:14134-45
Su, Yan; Egli, Martin; Guengerich, F Peter (2016) Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase ?. J Biol Chem 291:3747-56
Yeom, Mina; Kim, In-Hyeok; Kim, Jae-Kwon et al. (2016) Effects of Twelve Germline Missense Variations on DNA Lesion and G-Quadruplex Bypass Activities of Human DNA Polymerase REV1. Chem Res Toxicol 29:367-79
Su, Yan; Peter Guengerich, F (2016) Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases. Curr Protoc Nucleic Acid Chem 65:7.23.1-7.23.10
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 ?. J Biol Chem 291:24304-24313
Kim, Jae-Kwon; Yeom, Mina; Hong, Jin-Kyung et al. (2016) Six Germline Genetic Variations Impair the Translesion Synthesis Activity of Human DNA Polymerase ?. Chem Res Toxicol 29:1741-1754
O'Flaherty, D K; Patra, A; Su, Y et al. (2016) Lesion Orientation of O(4)-Alkylthymidine Influences Replication by Human DNA Polymerase ?. Chem Sci 7:4896-4904
Liu, Binyan; Xue, Qizhen; Tang, Yong et al. (2016) Mechanisms of mutagenesis: DNA replication in the presence of DNA damage. Mutat Res Rev Mutat Res 768:53-67
Patra, Amritaj; Zhang, Qianqian; Lei, Li et al. (2015) Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase ?. J Biol Chem 290:8028-38

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