Benzo(a)pyrene Mutagenic Mechanisms

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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).