Saccharomyces cerevisiae RAD52 alleles temperature-sensitive for the repair of DNA double-strand breaks

Abstract

We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to γ- radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, inter- chromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.