Translesion synthesis (TLS) DNA polymerases (Pols) help ensure the continued progression of the replication fork by promoting replication through DNA lesions. In the proposed studies, we will determine the roles of a number of human TLS Pols in promoting replication through a variety of DNA lesions induced by environmental pollutants and carcinogens and by cellular oxidative damage. In particular, we will test the hypothesis that replication through DNA lesions in human cells occurs via two distinct modes in which Pols, ?, ?, ?, Rev1, and ? mediate predominantly error-free TLS and act in a highly specialized manner dependent upon the DNA lesion, whereas Pol? performs lesion bypass in a more generalized and error-prone manner. Further, we will test the hypothesis that the more generalized and error-prone role of Pol? emanates from its ability to insert a purine nucleotide (nt), preferentially an A, opposite DNA lesions via a protein-template-directed mechanism. To elucidate the genetic bases of error-free and mutagenic replication through DNA lesions in humans, we will carry out the following studies.
In Aim 1, we will examine the contributions of various TLS Pols to error-free vs. mutagenic lesion bypass in human and mouse cells. The lesions to be studied include (6- 4) photoproduct induced by UV irradiation, 7,8-dihydro-8-oxogunaine (8-oxoG) generated from free-radical attack on guanine in DNA, 1,N6-ethenodeoxyadenosine (edA) generated from interaction of adenine with products of lipid peroxidation resulting from cellular oxidative damage and from exposure to chemical carcinogens, 1,N2-propano-2'-deoxyguanosine (PdG), a ring-closed form of acrolein generated from lipid peroxidation and which also is a ubiquitous environmental pollutant, and N2-dG adduct of the environmental carcinogen (+) anti-benzo[a]pyrene diol expoxide (BPDE).
In Aim 2, biochemical studies will be done to examine the proficiency of Pol? in synthesizing DNA opposite (6-4) TT photoproduct, 8-oxoG, ?dA, PdG, and N2-dG BPDE. By steady-state kinetic analyses we will determine the catalytic efficiency of Pol? for inserting nucleotides opposite the DNA lesion and for carrying out the subsequent extension reaction, and biochemical studies will be done to test the hypothesis that Pol? inserts a purine nt opposite DNA lesions via a protein- template-directed mechanism. The genetic and biochemical studies we propose here are highly relevant for delineating the roles of TLS Pols in promoting error-free vs. mutagenic lesion bypass during replication, and for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents in human cells. Our proposal for a predominantly error-free mode of TLS by Pols, ?, ?, ?, Rev1, and ? would predict a role for these Pols in cancer suppression, whereas a mutagenic mode of TLS by Pol? would predict a role for this Pol in enhancing genomic instability and carcinogenesis.

Public Health Relevance

DNA lesions are generated in human cells from cellular oxidative damage and from exposure to chemical and environmental carcinogens. The determination of roles of various DNA polymerases in promoting error-free vs. mutagenic lesion bypass during replication in human cells is important for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
4R01ES020833-05
Application #
8974412
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Shaughnessy, Daniel
Project Start
2012-01-27
Project End
2017-11-30
Budget Start
2015-12-01
Budget End
2017-11-30
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
Yoon, Jung-Hoon; Hodge, Richard P; Hackfeld, Linda C et al. (2018) Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct. J Biol Chem 293:2949-2958
Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong et al. (2017) Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine. J Biol Chem 292:18682-18688
Conde, Juan; Yoon, Jung-Hoon; Roy Choudhury, Jayati et al. (2015) Genetic Control of Replication through N1-methyladenine in Human Cells. J Biol Chem 290:29794-800
Yoon, Jung-Hoon; Park, Jeseong; Conde, Juan et al. (2015) Rev1 promotes replication through UV lesions in conjunction with DNA polymerases ?, ?, and ? but not DNA polymerase ?. Genes Dev 29:2588-602
Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong et al. (2014) A role for DNA polymerase ? in promoting replication through oxidative DNA lesion, thymine glycol, in human cells. J Biol Chem 289:13177-85
Yoon, Jung-Hoon; Acharya, Narottam; Park, Jeseong et al. (2014) Identification of two functional PCNA-binding domains in human DNA polymerase ?. Genes Cells 19:594-601