MMR-Coupled Translesion DNA Synthesis During Suppression of PAH-Induced Mutation

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DESCRIPTION (provided by applicant): Environmental polycyclic aromatic hydrocarbons (PAHs), when activated by cellular metabolism, form a variety of mutagenic and carcinogenic adducts with DNA. Cellular responses remain poorly characterized. The long- term goal of this dual-PI project is an understanding of the key cellular pathways that respond to PAH-derived DNA adducts - comprehensive enough to provide an information base for identification of individuals for whom PAH exposures particularly increase cancer risk. We hypothesize that the final net efficiencies and fidelities with which these interconnected pathways respond to specific DNA lesions determine their mutagenicity and carcinogenicity. Thus, risks of human exposure to PAHs depend on the particular adducts induced in DNA, and the genetically-determined potencies of responses to them in individuals. Our proposed exploratory research focuses specifically on coordination of bypass of PAH adducts by translesion DNA polymerases (TLS pols) with correction by mismatch-repair (MMR) of targeted and untargeted replication errors generated by such bypass. We will investigate MMR-dependent mutation-suppression and apoptosis endpoints in live animals treated with B[a]P-diol epoxide (B[a]PDE), and the biochemical pathway(s) leading to these endpoints. Specific Aim 1 is to determine the effects of MMR deficiency on B[a]PDE-induced genotoxicity in intact tissues in transgenic mice. Levels of stereospecific DNA adducts will be correlated to frequencies and spectra of mutations induced by B[a]PDE and to B[a]PDE effects on cellular turnover (cell replication and apoptosis) in the stem cell compartment of colonic crypts of transgenic Msh2-/- versus Msh2+/+. Specific Aim 2 is to use complex but biochemically accessible human nuclear extracts to analyze processing of novel model DNA substrates that contain excision-provoking base-mispairs (G/T) that are closely adjacent to template B[a]P- guanine adducts; such adducts must thus be bypassed by subsequent DNA resynthesis. Assays for the efficiency of bypass of adducts during post-MMR-excision DNA resynthesis will be developed, and fidelity of DNA resynthesis immediately downstream of the adducts will be analyzed by a novel method. The effects of modulation of the levels of TLS pols Pol: and Pol7 or of PCNA ubiquitination will be determined. The outcomes of the two interconnected aims will direct the hypotheses to be addressed in future comprehensive studies. PUBLIC HEALTH RELEVANCE: This exploratory work will provide the basis for more future more extensive studies. These will help identify individuals at increased risk for cancer induced by environmental agents.