Genetic and molecular characterization of proteins essential for DNA repair and recombination in Saccharomyces cervisiae

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9417573 Ramirez The primary objectives of this research starter grant are to clone and characterize the genes REC1 and REC3 of Saccharomyces cerevisiae that are thought function in the overlapping processes of mitotic recombination and DNA repair. rec1- and rec3- strains have been shown to be defective in mitotic recombination and possess temperature sensitive phenotypes rec 1- is a ts-lethal at 39{SYMBOL 176 \f "Symbol"}, while both rec1- and rec3- are ts with respect to mitotic recombination, X-ray sensitivity and sporulation at 36{SYMBOL 176 \f "Symbol"}. Additionally, both these mutations define distinct genes which map to chromosome 11. A yeast genomic library will be used to transform rec1- and rec3- strains of S. cerevisiae in order to clone the corresponding wild-type genes by complementation. Previous studies aimed at cloning the REC3 gene resulted in the isolation of a suppressor of rec3-, RPD3. (RPD3 is thought to encode pleiotropic transcriptional regulator encoded on chromosome XIV). Hence, we will focus our efforts on characterizing clones which complement the rec1- and rec3- mutations and which specifically hybridize to chromosome Vll DNA on a Southern blot of electrophoretically separated whole chromosomes. To accelarate cloning of REC3, we will also test a genetic clone that maps closely to the REC3 locus (i.e., a clone containing genetic material adjacent to the AR02 gene) to determine if it contains sequences which complement the rec3- defect. Once cloned, the REC1 and REC3 clones will be sequenced. The resulting genetic sequence will be compared to other sequences genetic sequence data bases to gain insight into the function of the proteins encoded by these genes. Cloned REC1 and REC3 DNA will also be used to create null mutations to provide definitive mapping information, to determine lethality, and to assay gene expression by Northern hybridization. Since we have found that RPD3 suppresses the rec3- defect, an additional objecti ve of this proposal is to determine the possible role played by RPD3 in recombination. This will be tested by testing the sensitivity of rpd- strains to ionizing radiation, methyl methane sulfonate (MMS), and measuring rates of recombination. %%% The information generated frorm, this basic research project will contribute to our knowledge of the regulation and/or enzymology of recombination and DNA repair in eukaryotes. ***