Many environmental carcinogens, both chemical and physical, cause cancer at least in part by producing mutations, as evidenced by characteristic exposure-related mutation spectra in human cancers and cancers associated with genetic defects in DNA repair, as well as animal models. The focus of the proposed continuing investigations on interaction of DNA polymerases with carcinogen-modified DNA will be structure-function relationships. One focus will be biochemical and in vivo studies with the DNA polymerases of a model organism, the crenarcheon Sulfolobus solfataricus, and approaches will be extended to human Y- family polymerases. (1) Structural and functional studies will be done on the four DNA polymerases of S. solfataricus, Dpo1, Dpo2, Dpo3, and Dpo4, including their roles in normal replication and past several DNA adducts, as well as defining the role of the heterotrimeric PCNA protein in this organism. Structures of these polymerases will be determined by X-ray crystallography (including PCNA complexes) and motions of individual parts will be analyzed using hydrogen-deuterium (H-D) exchange rates. A major goal is understanding trafficking of these four DNA polymerases at adduct-blocked replication forks, as a model for other systems. The in vivo functions of the polymerases will be determined using quantitation of proteins, transgenic knockouts, immunoprecipitation of polymerase partners, and site-specific mutagenesis of specific DNA adducts. (2) Analysis of microscopic events in catalysis by two model DNA polymerases will be done using rapid fluorescence kinetics (Dpo4 tryptophan mutants and a coumarin-labeled bacteriophage T7 polymerase mutant) and H-D exchange kinetics (Dpo4), with a view to events in incorporation opposite DNA adducts. (3) Structure-function relationships will be analyzed in recombinant human Y-family DNA polymerases using catalytically-active truncated versions of human pol 7, 9, and: using approaches developed with the Dpo polymerases, including X-ray crystallography, pre-steady-state kinetics, and H-D exchange kinetics. Collectively, these studies have the goal of extending the understanding of how important DNA polymerases catalyze correct and incorrect insertions and respond to DNA blocks, phenomena relevant to cancer.

Public Health Relevance

Many of the chemicals that cause cancer do so by binding to the DNA in the cells of the body. When this genetic material (damaged DNA) is copied by enzyme systems in the body called DNA polymerases, mistakes may occur (mutations) and lead to cancer. The goals of this project involve understanding how the DNA polymerases make such mistakes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES010375-13
Application #
8286306
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Shaughnessy, Daniel
Project Start
2000-08-05
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
13
Fiscal Year
2012
Total Cost
$416,146
Indirect Cost
$139,419
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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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 ι. J Biol Chem 291:21063-21073
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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 η. J Biol Chem 291:14134-45
Su, Yan; Egli, Martin; Guengerich, F Peter (2016) Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η. J Biol Chem 291:3747-56
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 η. J Biol Chem 291:24304-24313
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
Kotapati, Srikanth; Wickramaratne, Susith; Esades, Amanda et al. (2015) Polymerase Bypass of N(6)-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene. Chem Res Toxicol 28:1496-507
O'Flaherty, Derek K; Guengerich, F Peter; Egli, Martin et al. (2015) Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase η opposite Intrastrand Cross-Linked DNA. Biochemistry 54:7449-56

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