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 events in the yeast Saccharomyes cerevisiae. Many of the genes necessary for the repair of DNA double strand breaks (DSB) are required for the successful completion of the meiotic cycle suggesting that both processes proceed through similar molecular mechanisms. A central component for both the repair of DSBs and the successful completion of meiosis is the protein encoded by the RAD52 gene. In addition to its role in DSB repair and meiosis, RAD52 appears to influence spontaneous mitotic recombination. In strains carrying a complete deletion of the RAD52 gene mitotic recombination between repeats is reduced; however, this decrease is blocked by a concomitant mutation in the gene encoding the Rho-associated nuclease RhoNUC (RNC1). This result suggests that RNC1 is epistatic to RAD52 for spontaneous mitotic recombination. Interestingly rnc1 does not rescue the DSB repair deficiency or the meiotic lethality associated with rad52. 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". The elevated levels of "excision" seen in polIII or polI 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. RNC1 does not appear to play a role in this interaction since rnc1 rad52 polIII mutants are still blocked for Tn5 "excision".