The long-term goal of this project is to understand the molecular mechanisms of substrate specificity in two families of enzymes that play a critical role in the repair of ionizing radiation damage: the HhH-GPD Nth Superfamily and the Fpg Family. The DNA damages recognized by these oxidative DNA glycosylases have been implicated in the initiation of carcinogenesis. The fundamental hypothesis underlying the proposed work is that the structural framework provided by any glycosylase superfamily or thmily member can support a wide variety of specificities for the excised base after substitution of a small number of residues.
The specific aims are to determine the substrate specificities of a comprehensive set of members of the Nth Superfamily and Fpg Family of DNA glycosylases selected by phylogenetic/stmctural algorithms developed in Core A with the goal of identifying particular amino acid residues in the substrate binding pocket associated with recognition of specific substrates. Subsets of naturally occurring DNA glycosylases that have similar sequences but divergent substrate specificities, such as EcNth and Micrococcus luteus UV endonuclease (M1Uve) of the Nth SuperFamily as well as naturally occurring Fpg and Nei DNA glycosylases witl be examined. Specific amino acid residues in the substrate binding pockets of particular Nth Superfamity and Fpg Family members, targeted by Core A as involved in substrate recognition, will be altered and substrate binding properties examined. Standard steady state kinetic measurements will be determined with a set of oligonucleotide substrates that overlap those of all Nth Superfamily and Fpg Family members using a high throughput fluorescence plate reader in the Expression, Characterization and Crystallization Core. These biochemical studies wil] complement the structural studies in Project 2 and help target particular gtycosylases for crystallization. The combined resuks of Core A and Projects 1 and 2 should provide a sound basis for a better understanding of substrate specificity in these classes of DNA glycosylases.
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