MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

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Proteins binding to single-stranded DNA are expected to participate in DNA recombination and repair as well as DNA replication. Thus, three different single-stranded-DNA-binding proteins (SSB) have been purified from the yeast Saccharomyces cerevisiae and antibodies have been raised against them. Using the antibodies as probes, their genes have been identified and cloned from a Lambdagt11 yeast DNA library. Deletions of the genes were then constructed, the wild-type genes were replaced by the disrupted genes, and the resulting phenotypes were studied. 20kd SSB is essential for cell viability. However, C-terminal region of the 20kd SSB is still functional, but the cell is UV-sensitive. This strongly indicates that 20kd SSB is required for DNA repair and recombination as well as DNA replication. The RAD52 gene product is required for DNA recombination and repair in yeast. The gene has been cloned and its nucleotide sequence determined by other groups. However, this important gene product has not yet identified and purified. By aid of a computer we identified several possible antigenic regions in the RAD52 gene. The oligopeptides covering the antigenic regions were chemically synthesized and conjugated to BSA and antibodies were raised against the conjugates. In addition, several fusion plasmids of the RAD52 gene and either the LambdapL promoter or the yeast Alpha-mating type pheromon leader sequence or the yeast ADH promotor will be constructed in order to overproduce RAD52 protein in E. coli and yeast. Finally, one of the excision repair genes of yeast (RAD18), which also has been expected to be a subunit of yeast DNA polymerase I, has been cloned and characterized.