The x-ray sensitive mutants of the yeast Saccharomyces cerevisiae are a valuable genetic resource for understanding repair of ionizing radiation damage in eucaryotes. In yeast, double-strand DNA caused by x-rays and some chemical mutagens appear to be repaired by a recombinational mechanism, which probably involves both recombination between homologues and sister-chromatid exchange. Many genes including RAD50 to RAD57, are thought to be involved in recombinational repair and we have cloned several of these genes. We plan to continue existing projects aimed at characterizing repair of x-ray induced DNA damage in yeast, and the relationship between repair and recombination, using both Rad+ and Rad- strains. The various approaches proposed are different mechanistically, yet all address questions aimed at a better understanding of repair and recombination, and particularly the role of RAD genes in these processes. We will use our existing clones to continue a molecular and genetic analysis of the RAD24, RAD51, RAD54, RAD55 and RAD57 loci. We will also more thoroughly characterize mutations in RAD17 and RAD53, and clone these genes. RAD51 and RAD54 have been shown to be transcriptionally induced by DNA damage and RAD52 and RAD54 to be induced by meiosis; Northern hybridizations and lacZ fusion techniques will be used to continue studying the regulation of RAD51, RAD52 and RAD54. We will isolate from yeast the proteins encoded by RAD51, RAD54, RAD55 and RAD57, after first placing these genes under the control of a strong yeast promoter. We also plan to overexpress derivatives of our cloned genes in E. coli to produce fusion proteins which can then be used to raise antibodies. The antibodies will be used in the identification and purification of these proteins from yeast, and in studies of the subcellular localization of the RAD gene proteins. Isolated proteins will be tested for enzymatic activities such as ATPase, nuclease and DNA binding activities. We will use pulsed field gel electrophoresis in molecular studies of repair and recombination. We have developed a method that uses a circular derivative of a yeast chromosome to detect double-strand DNA breaks (dsb) and recombinant DNA molecules on pulsed-field gels. During meiosis, we can detect both sister-chromatid exchanges (SCE), and two types of molecules resulting from recombination between the circular chromosome and its linear homologue. We hope to detect similar molecules arising from repair in mitotic cells, and to characterize wild-type strains and study the molecular phenotypes of rad mutants in repair and meiosis. We hope to demonstrate a direct relationship between dsb and SCE after x-rays in Rad+ strains, and to determine whether rad mutants that abolish dsb repair also abolish SCE. Further meiotic experiments are also planned using this system, with the aim of examining at the molecular level both wild-type meiotic recombination and the severe meiotic defects of many of the rad mutations.
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