Many carcinogens become covalently attached to DNA and cause genotoxic damage by nucleotide misincorporation and polymerase blockage. Several lines of evidence indicate that these events are kinetically controlled and not governed only by thermodynamic stability of base pairing. Efforts in this laboratory are directed towards understanding interactions between DNA adducts and polymerases. HIV-1 reverse transcriptase (RT) will be crystallized with a 7,8-dihydro-8-oxoguanine (8-oxoG)-containing oligonucleotide and nucleoside triphosphate (dNTP), and results will be analyzed in the context of pre-steady-state kinetics. The kinetics of misincorporation and blocking of HIV-1 RT will be analyzed in terms of a model of multiple binary/ternary complexes vs. altered polymerase-DNA affinity, using 06-substituted guanines as a model. A series of N2-substituted guanine oligonucleotide derivatives will be used to systematically probe the effect of steric bulk on the kinetics of polymerase incorporation, blocking, and misincorporation. Other pre-steady- state kinetic studies will be directed towards using fluorescence and circular dichroism to directly observe putative conformational changes in polymerase cycles. Pre-steady-state kinetic approaches will be applied to polymerase delta, the major mammalian leading strand replicative enzyme, to determine the applicability of prokaryotic models in studies of normal incorporation, miscoding, and blockage by this enzyme. The general hypothesis is that normal incorporation, misincorporation, pausing, and complete blockage differ because these events represent a continuum of varying fits of polymerase- DNA-dNTP ternary complexes, reflected in rate constants for conformational changes and phosphodiester bond formation. The overall goal is understanding molecular mechanisms of mutagenesis as a part of chemical carcinogenesis.
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