DNA synthesis across unrepaired DNA damage, known as translesion DNA synthesis (TLS), is one of the major mechanisms responsible for chemical mutagenesis; in humans this process is associated with the development of cancer and age-related degenerative diseases. The goal of this research is to elucidate the mechanisms of TLS as they occur in human cells. Several TLS-specialized DNA polymerases (pol), such as pol h, pol k, pol i, pol z and REV1, have been discovered recently; we will determine the role of these enzymes in TLS in human cells. Two exocyclic propanodeoxyguanosine (PdG) DNA adducts and 1,N6-ethenodeoxyadenosine (edA) will be employed as target lesions. We have established the genotoxicity of these adducts in human cells, using a unique shuttle vector system developed in my laboratory. To probe the mechanism for TLS in cells, primer extension studies with purified polymerases will be first conducted to determine their efficiency, fidelity, and coding specificity in vitro. By comparing in vivo and in vitro results, candidate polymerase(s) responsible for TLS events in cells will be identified. The expression of the candidate polymerase gene(s) in human cells will be silenced by RNA interference technology. Such """"""""engineered"""""""" cells are then used as hosts for genotoxic analyses. By comparing TLS events in """"""""wild type"""""""" and engineered cells, the role and function of candidate polymerase(s) in TLS can be assessed. Extracts of engineered cells will be used to replicate modified plasmid in vitro for biochemical studies. The combination of in vitro TLS studies, in vivo assays for genotoxicity, and RNA interference technology will provide novel insights into the fundamental role and function(s) of TLS polymerases and their contribution to chemical mutagenesis in human cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076163-08
Application #
7190048
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1999-08-15
Project End
2009-07-31
Budget Start
2007-04-01
Budget End
2009-07-31
Support Year
8
Fiscal Year
2007
Total Cost
$214,052
Indirect Cost
Name
State University New York Stony Brook
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Hashimoto, Keiji; Cho, Youngjin; Yang, In-Young et al. (2012) The vital role of polymerase ? and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ? by REV1 to replication-stalled site. J Biol Chem 287:9613-22
Yang, In-Young; Hashimoto, Keiji; de Wind, Niels et al. (2009) Two distinct translesion synthesis pathways across a lipid peroxidation-derived DNA adduct in mammalian cells. J Biol Chem 284:191-8
Kalam, M Abul; Haraguchi, Kazuhiro; Chandani, Sushil et al. (2006) 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 34:2305-15
Pollack, Michael; Yang, In-Young; Kim, Hye-Young H et al. (2006) Translesion DNA Synthesis across the heptanone--etheno-2'-deoxycytidine adduct in cells. Chem Res Toxicol 19:1074-9
Stein, Scott; Lao, Yanbin; Yang, In-Young et al. (2006) Genotoxicity of acetaldehyde- and crotonaldehyde-induced 1,N2-propanodeoxyguanosine DNA adducts in human cells. Mutat Res 608:1-7
Hashimoto, Keiji; Tominaga, Yohei; Nakabeppu, Yusaku et al. (2004) Futile short-patch DNA base excision repair of adenine:8-oxoguanine mispair. Nucleic Acids Res 32:5928-34
Yang, In-Young; Miller, Holly; Wang, Zhigang et al. (2003) Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells. J Biol Chem 278:13989-94
Yang, In-Young; Chan, Grace; Miller, Holly et al. (2002) Mutagenesis by acrolein-derived propanodeoxyguanosine adducts in human cells. Biochemistry 41:13826-32
Yang, In-Young; Johnson, Francis; Grollman, Arthur P et al. (2002) Genotoxic mechanism for the major acrolein-derived deoxyguanosine adduct in human cells. Chem Res Toxicol 15:160-4
Yang, I Y; Hossain, M; Miller, H et al. (2001) Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli. J Biol Chem 276:9071-6

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