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 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 Lambdagtll yeast DNA library. Deletions of these genes were then constructed, the wild-type genes were replaced by the disrupted genes, and the resulting phenotypes were studied. 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, an in vitro DNA recombination system yeast Delta-Delta sequences has been developed. This system requires ATP, MgCl2, and supercoiled plasmid DNA containing at least two delta sequences; it generates a double-strand break near or at the one of delta sequences but does not cleave plasmid DNA lacking delta sequences.