Our hypothesis is that defects in the enzyme families we study result in aberrant base excision and homology-directed repair which is an engine driving human carcinogenesis. The majority of endogenous and radiation-induced DNA lesions are removed by the base excision repair (BER) machinery and when this pathway fails, the resulting substrates are channeled into homology-directed repair. The overall goals of this Program Project are to understand at the atomic level how three families of DNA repair enzymes the HhH-GPD superfamily of DNA glycosylases, the Fpg/Nei family of DNA glycosylases and the RecA-RAD51 family of recombinases, recognize and process their substrates and how germ line and tumor associated variants of these proteins influence cancer susceptibility and carcinogenesis, respectively. In order to translate our basic science more directly to cancer, we now propose to use our expertise and tested methodologies to examine human genetic variation. Based on our discoveries of novel substrate specificities and biochemical activities, as well as our strengths in fundamental biochemistry and structural biology, our program for the renewal will be informed and driven by the identification and characterization of germ line and tumor-associated variants of human base excision repair and homology-directed repair enzymes. Core A will identify human germ line and somatic DNA sequence variants of the oxidative DNA glycosylases and RAD51 based on structure and phylogeny. Project 1 will demonstrate whether these repair variants Induce cellular transformation, are mutagenic in mouse cells and whether they influence the cellular response to ionizing radiation and chemotherapeutic agents. Project 2 will examine the biochemical properties of the oxidative glycosylase variants and solve structures of wild type enzymes with substrates and where appropriate the glycosylase variants. Project 3 will examine the biochemical and where appropriate, structural characteristics of RAD51 variants as well as study the mechanisms of RAD51 filament formation. Project 4 will reconstitute the base excision repair pathway in the context of nucleosomes with wild type and variant glycosylases and examine the effect of histone primary sequence variants on chromatin accessibility during BER. Projects 1-4 will be serviced by the Protein and Biochemistry Core B which will supply purified proteins and perform high throughput analysis of the proteins. In addition to bioinformatics for all projects, Core A will also perform kinetics analysis for Projects 2-4. Core C will provide the administrative underpinnings for the project.
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