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 BER and when this pathway fails, the resulting substrates are channeled into HDR. The overall goals of this Program Project are to understand at the atomic level how three families of DNA repair enzymes, the HhH and Fpg/Nei families 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. Based on our strengths in biochemistry and structural biology, our program is now informed and driven by the identification and characterization of germ line and tumor-associated variants of human base excision repair and HDR 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. Project 5 will define at the single molecule level the search mechanisms of the BER and HDR enzymes and their variants. Projects 1-5 will be serviced by Core B which will supply purified proteins and perform high throughput analysis of the proteins. Core A will perform bioinformatics analysis for all projects and kinetics analysis for Projects 2-4
These studies will provide insight into how the BER and RAD51 variants in the normal population and in tumors may contribute to altered DNA repair capacity. Moreover, this aberrant DNA repair in tumor cells may be a key cause of genetic instability in cancer that leads to tumor progression. Our approach may also offer the possibility of devising therapeutic strategies to which tumors that express DNA glycosylase and RAD51 variants are susceptible.
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