The overall goal of the Program Project Structure, Function and Evolution of DNA Repair Enzymes is to understand the fundamental mechanisms underpinning the Nth Superfamily and the Fpg Family of DNA glycosylases and the RecA recombinases, enzymes that process ionizing radiation-induced DNA damages, damages known to initiate the carcinogenic process. The central hypothesis underpinning this Program Project is that the families of the DNA repair enzymes in question each have a structural framework that supports a variety of significant changes in specificity or regulatory properties with only a small number of sequence alterations. To test this hypothesis a novel phylogenetic/structural analysis will be used to develop algorithms not only to identify natural protein variants that are orthologous, but have different activities, but also to determine which amino acids in a particular protein should be varied to potentially alter substrate specificity or other protein functions. Since the DNA glycosylases that initiate Base Excision Repair and the RecA recombinases involved in Double Strand Break Repair are highly conserved across all three kingdoms, they are particularly suited to this approach. The proposed Program Project consists of three Projects and three Cores. Core A, the Bioinformatics Core, will use a number of methodologies to select the proteins to be examined in the projects to test our hypothesis. Project 1 is designed to delineate and alter the substrate specificities of the oxidative DNA glycosylases targeted by Core A. Project 2 will focus on particular DNA glycosylases and determine their crystal structures. Project 3 will use similar computational, biochemical and structural approaches to understand variations in the biochemical properties of the RecA recombinases. The projects directly depend on Core A to provide the analysis of the natural protein sequences and to provide support for experimental design and will test the hypotheses derived so that there is continued iteration among the projects. All three projects will be supported by the Expression, Characterization, and Crystallization Core (Core B) and the Administrative Core (Core C).
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