MECHANISMS OF HOTSPOT MUTAGENESIS BY (+)-BPDE

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The objective of the proposed research is to reveal fundamental relationships between chemical structure and mutagenic/carcinogenic activity, and to provide insight into the molecular mechanisms by which carcinogens after DNA, cause mutations and result in cancer. Using several site-specifically adducted oligonucleotides, we will compare how the structure, conformation and position of the DNA adduct affect the fidelity, processivity and efficiency of DNA polymerases in vitro. We will also utilize these oligonucleotides in an in vivo model to analyze how these characteristics of an adduct affect the induced mutation. These oligomers contain the exact sequence context of two in vivo hotspots for (+)-BPDE (an ultimate carcinogenic metabolite of benzo[a]pyrene) and have the (+)-BPDE modification on the purine base either at or next to the hotspot. Nonadducted and (-)-BPDE (a weak or noncarcinogenic enantiomer) adducted oligomers will also be prepared for comparative studies. The specific aims are to: 1. Study the mutagenic effects of site-specific adducts by the primer extension reaction with the replicative DNA polymerases from human and the lesion bypass polymerase from yeast. We will a) Prepare site-specifically adducted 59- mer templates. b) Determine the effects of BPDE adduction on the fidelity, processivity and efficiency of DNA polymerases. c) Compare the effects of BPDE stereochemistry on DNA polymerase activities. d) Investigate effects of adduct position on DNA polymerase activities. 2. Study the mutagenic effects of site-specific adducts in vivo by transfection assays in mammalian cells. We will a) Construct several eukaryotic expression vectors so that each contains the entire coding region of the hprt gene and a single site-specific (+)- or (-)-BPDE adduct at or next to a hotspot in the hprt cDNA sequence. b) Determine the mutagenic effect of the site-specific adduct in vivo by transfecting these hprt expression vectors into V-79 cells. We will then analyze mutations in stable transformants and compare them with mutations obtained from our earlier random mutagenesis studies in V-79 cells. We will subsequently examine how the adduct position and chirality affect the outcome of the mutation formation.