MOLECULAR MECHANISMS OF DNA REPAIR AND RECOMBINATION IN YEAST

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We are investigating the unique chromosomal metabolic events associated with meiosis and the repair of chromosomes following exposure to DNA damaging agents. Many of the genes necessary for the repair of DNA double strand breaks (DSB) are required for the successful completion of the meiotic cycle. In particular the RAD52 gene is essential for the repair of DSBs and the successful completion of meiosis. Furthermore, RAD52 appears to influence mitotic recombination. To investigate the role of a RAD52, we have created strains containing a chromosomal resident deletion of RAD52 and a RAD52 gene under the control of the yeast GAL1 promoter. This allows us to tightly regulate the expression of the RAD52 gene. We have found that for the repair of DSBs the presence of RAD52 protein is required prior to treatment with ionizing radiation. Interestingly, the presence of a previously isolated nuclease (RhoNUC) appears to be influenced by RAD52, however, this control appears to be post-translational. In order to identify other proteins which may interact with RAD52 we have begun screening a "protein interaction" library. The possible role of RAD52 during replication is also being investigated. Using a unique system whereby we measure the frequency of "excision" of a bacterial transposon Tn5 placed into the yeast LYS2 gene, we have found that mutations in the gene encoding DNA polymerase I or III lead to high levels of Tn5 "excision". No increase is observed in DNA polymerase II mutants. The elevated levels of "excision" seen in po1III or po1I strains is reduced when a null mutant of RAD52 is introduced into these strains. This suggests an interaction between the enzymes proposed to be responsible for DNA lagging strand synthesis and RAD52.