Our long-term objective is to understand mechanisms of lesion bypass in eukaryotes. Lesion bypass is an important cellular response to unrepaired DNA damage during replication. Error-prone lesion bypass is the major mechanism of DNA damage-induced mutagenesis and represents a major factor in carcinogenesis. In eukaryotes, mechanisms of lesion bypass are poorly understood. Lesion bypass requires a specialized DNA polymerase (Pol). However, it is not clear which polymerases are involved in lesion bypass in cells. We hypothesize that (a) Polzeta, Poln, Polkappa, and Rev1 are involved in DNA synthesis opposite the lesion in vivo; and (b) efficient bypass of some lesions in cells requires nucleotide incorporation opposite the lesion by one polymerase and subsequent extension DNA synthesis by Polzeta. Polzeta , Poln, Polkappa, and Rev1 are able to incorporate nucleotides opposite several DNA lesions in vitro. In this proposal, the role of these polymerases in lesion bypass in vivo and their relationships to damage-induced mutagenesis will be studied.
Our specific aims are: (1) to study the function of Polzeta in DNA lesion bypass; (2) to determine the role of Rev1 in DNA lesion bypass; (3) to investigate cellular function of Poln in response to chemical induced DNA lesions; and (4) to examine the in vivo role of Polkappa in DNA lesion bypass and its relationship to damage-induced metagenesis. To accomplish the aims, in vivo genetic experiments will be performed using the yeasts S. cerevisiae and S. pombe as eukaryotic model organisms. In vivo mutagenesis experiments will be performed in yeast strains proficient or deficient in polzeta, Poln, Po1kappa, and Rev1 activities, using-stranded plasmid vector containing a site-specific DNA damage, including abasic site and four stereoiomeric anti-benzo[a]pyrene-N2-dG adducts in several sequence contexts. Genetic results will be interpreted with the help of in vitro biochemical experiments. The proposed studies should significantly advance our understanding of mechanisms of lesion bypass and damage-induced mutagenesis in eukaryotic cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Chemical Pathology Study Section (CPA)
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Okano, Paul
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University of Kentucky
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Zhou, Ying; Wang, Jillian; Zhang, Yanbin et al. (2010) The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis. Nucleic Acids Res 38:5036-46
Shachar, Sigal; Ziv, Omer; Avkin, Sharon et al. (2009) Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals. EMBO J 28:383-93
Wang, Zhigang (2006) Controlled expression of recombinant genes and preparation of cell-free extracts in yeast. Methods Mol Biol 313:317-31
Zhao, Bo; Wang, Jillian; Geacintov, Nicholas E et al. (2006) Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells. Nucleic Acids Res 34:417-25
Xie, Zhongwen; Zhang, Yangbin; Guliaev, Anton B et al. (2005) The p-benzoquinone DNA adducts derived from benzene are highly mutagenic. DNA Repair (Amst) 4:1399-409
Guo, Dongyu; Xie, Zhongwen; Shen, Huiyun et al. (2004) Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein. Nucleic Acids Res 32:1122-30
Zhao, Bo; Xie, Zhongwen; Shen, Huiyun et al. (2004) Role of DNA polymerase eta in the bypass of abasic sites in yeast cells. Nucleic Acids Res 32:3984-94
Xie, Zhongwen; Braithwaite, Elena; Guo, Dongyu et al. (2003) Mutagenesis of benzo[a]pyrene diol epoxide in yeast: requirement for DNA polymerase zeta and involvement of DNA polymerase eta. Biochemistry 42:11253-62
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
Zhang, Yanbin; Wu, Xiaohua; Guo, Dongyu et al. (2002) Lesion bypass activities of human DNA polymerase mu. J Biol Chem 277:44582-7

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