The human population is exposed to a variety of environmental cancer-causing pollutants that include fossil fuel combustion products and substances in cigarette smoke. The bay region benzo[a]pyrene (B[a]P) is the best known representative of a class of potentially carcinogenic compounds, the polycyclic aromatic hydrocarbons (PAH). Like other PAH substances, B[a]P is metabolically activated to highly reactive and mutagenic B[a]P dial epoxides that react with DNA forming adducts. Inefficient DNA repair and translesion synthesis of DNA adducts catalyzed by polymerases, especially the recently discovered bypass polymerases, are key factors that determine if a bulky lesion can give rise to mutations and ultimately to cancer. However, the molecular bases of these biologically important phenomena are still poorly understood. The major objectives of this project are to elucidate (1) the mechanisms by which human DNA repair enzymes recognize and excise bulky DNA adducts such as those derived from B[a]P diol epoxides, and (2) the molecular-structural factors and mechanisms involved in translesion synthesis catalyzed by representative bypass polymerases (Dpo4, pol kappa, and pol eta) in vitro. These questions are addressed using well defined DNA sequences with site-specifically incorporated lesions derived from the binding of B[a]P diol epoxides to the exocyclic amino groups of adenine (dA) and guanine (dG) in DNA.
Specific Aim I. Determine the DNA structural factors and adduct conformations that cause efficient or inefficient nucleotide excision repair of stereochemically defined B[a]P-dG and B[a]P-dA lesions employing variable base sequence context as a tool to modulate the local structural properties of the DNA.
Specific Aim 2. Determine and compare the differences and mechanisms involved in the fidelity and efficiency of translesion bypass of bulky B[a]P-dG and B[a]P-dA lesions in different base sequence contexts by Y family bypass polymerases. Modern computational and modeling techniques, based on NMR structural studies, will be employed to derive insights into adduct conformational properties and specific DNA distortions in the vicinity of the adducts that serve as signals of recognition and removal of the lesions by nucleotide excision repair proteins. Molecular dynamic simulation methods will be employed to investigate base sequence and adduct stereochemistry-dependent translesion bypass with the lesions positioned in different base sequence contexts that are known to modulate translesion synthesis.
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