DNA repair systems identified in mitotic cells of the yeast Saccharomyces cerevisiae are being examined for their protection of cells undergoing meiosis and the role of the corresponding genes in normal meiosis. the RAD50, RAD52, and RAD57 genes are essential in the repair of DNA double-strand breaks in mitotic cells. We have shown that they are also required for meiosis. Mutations abolish normal meiotic recombination; RAD50 acts early in meiosis. Rare single-strand interruptions (SSIs) are observed in rad52 and rad57 strains which appear to be related to recombination. Gentle isolation techniques have allowed the characterization of SSIs as breaks in DNA; many have 3' OH and 5' PO4 termini. The SSIs do not appear to be randomly distributed, suggesting specific sites or regions involved in normal meiotic recombination. We are investigating the distribution of the breaks using complete sets of fragments of entire chromosomes. DNA is gently isolated, restricted, and displayed using alkaline agarose gel electrophoresis. The gels are probed with individual fragments to determine if the rare breaks seen with alkaline sucrose gradients can be identified within specific sequences of the chromosome. The rad52 and rad57 mutants are concluded to be defective in meiotic recombination; however recombinants can be recovered prior to commitment to reductional division. The frequency in rad52 is much lower than in Rad+ strains, but it is comparable in rad57 mutants. The recombinants differ qualitatively from those in Rad+. When meiosis is arrested and rad52 and rad57 are exposed to growth medium, recombinants are not recovered. This is due to the extended time necessary for recombinants to form, suggesting that rad52 and rad57 are blocked at an intermediate step. The rad57-1 temperature sensitive allele has allowed us to investigate this more extensively and determine the timing of events. Both single-strand and double strand interruptions are found in rad57 mutants. Based on genetic and biochemical changes, the order to gene function appears to be RAD50, RAD52, and RAD57.
