Polycyclic aromatic hydrocarbons (PAHs) are pervasive in the environment, arising during combustion processes. Many of these PAHs have been demonstrated to be carcinogenic, following metabolic activation by cytochrome P450s. Environmental exposure to PAHs results in the formation of numerous types of DNA lesions, but their individual contributions to mutagenesis and carcinogenesis are largely unknown. In order to provide si - and stereospecifically PAH modified oligonucleotides to determine the mechanisms and consequences of DNA bypass replication of these lesions. These findings will be correlated with DNA adduct structures determined by NMR. The biological fate of DNAs containing specific PAH lesions will be defined by the intrinsic activities associated with the particular polymerase that encounters the lesion and multiple factors associated with the identity and geometry of the adduct base. This objective will be accomplished through four, multifaceted specific aims. 1) Kinetic analyses of replication past site- and stereospecifically modified DNA bases. Using gel fidelity assays with a variety of polymerases, we will establish individual rate constants along the reaction pathway as they are affected by various adduct geometries; 2) Mutagenic frequency and spectrum of PAH-adducted DNAs resulting from translesion synthesis in human cell extracts. To understand how human replicative polymerases process site- and stereospecific PAH adducts, the modified oligonucleotides will be engineered into vectors that will permit analyses of fidelity in both leading and lagging strand synthesis; 3) Relationship of error-prone bypass replication and the presence of mismatched bases near the adduct site. The biochemical nature of error prone replication past DNA lesions in the presence of mismatched bases and its importance in mutagenesis and carcinogenesis will be investigated; 4) Structure-activity relationship among various bay- and nonbay-region PAHs and their relative levels of lethality an mutagenesis. A series of ba versus nonbay-region PAH adducts will be evaluated ina single-stranded DNA vector system for their relative levels of lethality and mutagenesis.
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