The potent environmental carcinogen benzo[a]pyrene (B[a]P) is metabolically activated in cells to (+)-anti- B[a]PDE, which forms one primary adduct: [+ta]-B[a]P-N2-dG. [+ta]-B[a]P-N2-dG induces different mutational patterns depending on sequence context (e.g., >95% G->T vs. 95% G->A in 5'-TGC vs. 5'-AGA sequences). Evidence suggests that these different mutations arise from different adduct conformations (as influenced by sequence context), when bypassed by different DNA polymerases. For [+ta]-B[a]P-N2-dG, we showed that E. coli DNA Pol V is involved in dATP bypass (G->T mutations), while dCTP insertion (no mutation) involves Pols IV and V. With the mirror image adduct [-ta]-B[a]P-N2-dG, Pol V does dATP insertion, while Pol IV alone is required for dCTP bypass. Literature findings suggest that in general DNA Pol V has two modes of adduct bypass: (1) correct dNTP insertion, and (2) default dATP insertion. Understanding the mechanism of correct vs. mutagenic insertion is hampered by no X-ray structure for UmuC, which is the the polymerase subunit of DNA Pol V. Using homology modeling, we constructed a UmuC model, which revealed active site amino acids potentially involved in dictating dNTP insertion. Active site amino acids were changed. In cells we showed that mutant-UmuCs could increase (up to ~10-fold), or decrease (~5-fold) dATP insertion compared to wt-UmuC. The goal of this project is to understand what amino acid residues define correct (dCTP) vs. incorrect (dATP) insertion for Pols IV and V and how these pathways are controlled by the cell. Studies in cells and in vitro with mutant and wild type Pols IV and V are proposed. Literature findings show that Pol IV is equivalent to human Pol k, while Pol V is equivalent to human Pol h.
Aim 1. Establish the roles of Pol IV vs. Pol V in cells;i.e., which does insertion vs. extension.
Aim 2 : Determine what amino acids in Pols IV and V control correct (dCTP) vs incorrect (dATP) insertion;e.g., why does Pol IV do correct (dCTP) insertion, while Pol V does incorrect (dATP) insertion, with [-ta]-B[a]P-N2-dG.
Aim 3 : Determine what lesion-bypass Pols are involved in G->A mutagenesis (dTTP insertion).

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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Boston University
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Sholder, Gabriel; Creech, Amanda; Loechler, Edward L (2015) How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene. DNA Repair (Amst) 35:144-53
Sholder, Gabriel; Loechler, Edward L (2015) A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh. DNA Repair (Amst) 25:97-103
Ikeda, Mio; Furukohri, Asako; Philippin, Gaelle et al. (2014) DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts. Nucleic Acids Res 42:8461-72
Chandani, Sushil; Loechler, Edward L (2013) Structural model of the Y-Family DNA polymerase V/RecA mutasome. J Mol Graph Model 39:133-44
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Chandani, Sushil; Loechler, Edward L (2009) Y-Family DNA polymerases may use two different dNTP shapes for insertion: a hypothesis and its implications. J Mol Graph Model 27:759-69
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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

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