Mutagenic and Repair Mechanisms of Endogenous DNA Damage

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The long term goal of this project is to elucidate molecular mechanisms of mutagenesis, translesion DNA synthesis (TLS) and base excision repair, correlating these biological findings with the three-dimensional structure and thermodynamic properties of oxidatively damaged DNA. The specific aims are (a) to establish the pathway(s) of translesion synthesis past oxidatively damaged DNA in human cells, using a novel experimental system to quantify the efficiency and fidelity of this process (b) to identify, among the myriad of recently discovered DNA polymerases, those enzymes specifically engaged in replicative and repair translesion syntheses in human cells and (c) to identify amino acid residues that participate in recognition of DNA damage and in determining substrate specificity. This research will focus on major forms of oxidative damage found endogenously in DNA, including thymine glycol, formamidopyrimidines, exocyclic DNA adducts and 8-oxoguanine. A novel shuttle vector system has been developed that will allow us to explore translesion synthesis events in human cells. This quantitative system measures the efficiency, fidelity and coding properties of lesions undergoing translesion synthesis and will be used to establish the genotoxicity of damaged DNA bases. RNA interference technology will be used to explore the role and function(s) of translesion synthesis-specialized DNA polymerases in human cells. Structural information on DNA glycosylases, obtained by x-ray crystallography, will be combined with insights gained from bioinformatics and molecular modeling methods to explore mechanisms of DNA damage recognition during base excision repair. These studies provide significant insights into the molecular biology of translesion synthesis, the central event in miscoding by DNA polymerases, and into several functions of DNA glycosylases, enzymes that initiate DNA repair. As such, this research forms the biologic focus for projects 2, 3 and 4.