A GENERAL-METHOD FOR IDENTIFYING RECESSIVE DIPLOID-SPECIFIC MUTATIONSIN SACCHAROMYCES-CEREVISIAE, ITS APPLICATION TO THE ISOLATION OF MUTANTS BLOCKED AT INTERMEDIATE STAGES OF MEIOTIC PROPHASE AND CHARACTERIZATION OF A NEW GENE SAE2

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that co nfer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a def ect in spore formation that is dependent upon a gene required for init iation of meiotic recombination and development of meiosis-specific ch romosome structure (SPO11). Identified in this screen were null mutati ons of the DMC1 gene, nonnull mutations of RAD50 (rad50S), and mutatio ns in three new genes designated SAE1, SAE2 and SAE3 (Sporulation in t he Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutant s are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previousl y identified nonnull mutations of RAD50 (rad50S). Most notably, both m utations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover o f meiosis-specific double-strand breaks. These observations provide fu rther evidence that this constellation of phenotypes identifies a spec ific molecular function.