Distinct mechanisms have evolved to repair DNA damaged by ultraviolet and ionizing radiations, by chemicals, and by reactive biological molecules generated during cellular metabolism. Failure to repair damaged DNA invariably results in increased cell death and a hypermutational phenotype, and may lead to carcinogenesis in multicellular organisms due to the elevated genetic mutation load. The mechanism by which eukaryotes repair chromosomal DNA breakages induced by ionizing radiation remains obscure at the molecular level, although there is evidence that the DNA strand break repair system is also required for the successful completion of V(D)J recombination in mammals. Recent molecular cloning studies have demonstrated the evolutionary conservation of the DNA strand break repair machinery from the yeast Saccharomyces cerevisiae to mammals, indicating that information garnered from studies in S. cerevisiae will be invaluable for delineating the action mechanism of the equivalent repair system in humans. Characterization of the S. cerevisiae genes -RAD5O, RAD51, RAD52, RAD53, RAD54, RAD55, RAD56, and RAD57 - required for DNA strand break repair has revealed that they also mediate meiotic and mitotic genetic recombination. Existing evidence suggests that RAD51, RAD52, and RAD54 proteins carry out biochemical reactions essential for recombination and the repair of DNA strand breaks, while the remaining gene products act to increase the efficiency of these reactions. The RAD51 protein bears structural homology to the Escherichia coli RecA protein and appears to physically interact with RAD52 protein. The RAD51 protein has been overproduced in S. cerevisiae and purified to homogeneity in this laboratory. RAD51 protein has DNA dependent ATPase activity, and importantly, it catalyzes the ATP-dependent pairing of homologous DNA molecules and DNA strand exchange, which are central biochemical reactions in genetic recombination and the recombinational repair of DNA strand breaks. The interaction of RAD51 with ATP and DNA will be characterized by nitrocellulose filter binding and by isolating RAD51/ligand complexes using molecular sizing columns. The homologous DNA pairing and strand exchange activities will be examined using in vitro recombination systems that involve two, three or four DNA strands. The self association properties of RAD51 will be examined using molecular sizing columns and glycerol gradient centrifugation. The biological significance of these RAD51 activities will be addressed by hydroxylamine mutagenesis and site- directed mutagenesis. The RAD52 protein has been overproduced in S. cerevisiae. RAD52 will be purified to homogeneity and its biochemical properties examined. The functional interplay between RAD51 and RAD52 proteins in the context of homologous DNA pairing and strand exchange will be examined. These proposed studies represent the starting point in dissecting the molecular mechanism of homologous recombination and repair of chromosomal breakage in eukaryotes.
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