Many carcinogens become covalently attached to DNA and cause genotoxic damage due to polymerase blockage or nucleotide misincorporation. These events are controlled kinetically by the individual DNA polymerases. Continued studies are proposed on the interaction of a series of carcinogens, bound to oligonucleotides, with a set of both replicative and so-called translesion bypass polymerases, including viral (DNA polymerase T7-, HIV-1 reverse transcriptase) and bacterial (Dpo4) model polymerases as well as recombinant human polymerases (delta, eta, iota, kappa, REV1). Kinetic analyses will be done with these polymerases and the 22 DNA-carcinogen adducts available, varying in size from oxygen and methyl groups to large polycyclic hydrocarbons, identifying changes in rate-limiting events related to normal incorporation vs. misincorporation and blocking. The working hypothesis is that alternate conformations and inactive complexes are important, and some of these may also have rapid nucleotide dissociation kinetics. Another use of kinetic analysis will be to understand the chronology and coupling of the events involved in changing polymerases at DNA damage sites, i.e. between replicative and translesion polymerases. Roles of accessory proteins will be considered. Pre-steady-state kinetic spectroscopic approaches will also be used to better define events related to individual steps of polymerase catalysis, with fluorescence and circular dichroism changes being compared to measured rates of product formation. Work on the crystallography of carcinogen-adducted DNA with polymerases (HIV-1 reverse transcriptase, Dpo4) is in progress and will be expanded, with the goal of linking temporal events with specific structural changes. The general working hypothesis is that normal incorporation, misincorporation, pausing, and blockage represent a continuum of events related to fits and rates of reactions involving polymerase-DNA-dNTP ternary complexes, and that these can be understood in quantitative terms (rate constants and structures). The overall goal is understanding molecular mechanisms of mutagenesis and relevance to chemical carcinogenesis. ? ?
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