MOLECULAR PHARMACOLOGY OF TUMOR AND VIRUS INHIBITORS

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This research focusses on the biological consequences of DNA damage with the overall goal of elucidating primary events in carcinogenesis. A principal theme is to establish relationships between the structure of damaged DNA and the functions of enzymes involved in DNA replication and repair. Novel experimental systems have been developed for this purpose. Site specific mutagenesis involves a strategy in which a shuttle plasmid vector, containing a single defined lesion, is allowed to replicate in mammalian cells or bacteria. The position of mutations induced is established by DNA sequence analysis. Primer-extension reactions, catalyzed by DNA polymerase, coupled with steady-state kinetic analysis, are used to explore translesional synthesis and mutagenic events in vitro. Our specific aims are (a) to establish models for frameshift mutagenesis in terms of misaligned DNA templates and kinetics governing translesional synthesis; (b) to elucidate the molecular basis underlying sequence context effects on base substitutions and deletions; (c) to understand the role of SOS functions in translesional synthesis; (d) to discover pathways by which mutations are generated during repair of bistrand abasic sites in DNA; (e) to demonstrates differences between DNA polymerases in their abilities to generate mutations arising from DNA damage; (f) to develop in vitro assays that predict mutagenic specificity for defined DNA lesions in vivo; (g) to explore mechanisms by which DNA damage enhances the frequency of homologous recombination in mammalian cells and bacteria; and (h) to establish the solution structure of misaligned intermediates formed during deletion mutagenesis. Additional studies are designed (a) to determined the substrate specificity of Fpg protein; (b) to establish the role of the N-terminus in the catalytic function of this enzyme; (c) to reveal the structural basis for binding of the zinc finger domain oxidatively-damaged DNA; (d) to elucidate a catalytic mechanism for DNA glycosylate activity; (e) to detect functional groups on Fpg protein and its substrates that facilitate "recognition" of oxidative damage; (f) to establish the structure of complexes formed between Fpg protein or adenine DNA glycosylate and analogs of their DNA substrates; and (g) to study substrate binding and mechanism of action of selected AP endonucleases, and (h) to quantify the contribution of Fpg protein to DNA repair in cells.