An allele of RFA1 suppresses RAD52-dependent double-strand break repair inSaccharomyces cerevisiae

An allele of RFA1, the largest subunit of the single-stranded DNA-binding c omplex RP-A, was identified as a suppressor of decreased direct-repeat reco mbination in rad1 rad52 double mutants. In this study, we used two LEU2 dir ect-repeat assays to investigate the mechanism by which the rfa1-D228Y alle le increases recombination. We found that both intrachromatid and sister ch romatid recombination are stimulated in rfa1-D228Y strains. In a rad1 rad52 background, however, the majority of the increased recombination is caused by stimulation of deletion events by an intrachromatid recombination mecha nism that is likely to be single-strand annealing. Studies in which an HO e ndonuclease cut was introduced between the two leu2 copies indicate that th e rfa1-D228Y mutation partially suppresses the rad52 defect in recovering r ecombination products. Furthermore, molecular analysis of processing and pr oduct formation kinetics reveals that, in a rad52 background, the rfa1-D228 Y mutation results in increased levels of recombinant products and the disa ppearance of large single-stranded intermediates characteristic of rad52 st rains. On the basis of these results, we propose that in the absence of wil d-type Rad52, the interaction of RP-A with single-stranded DNA inhibits str and annealing, and that this inhibition is overcome by the rfa1-D228Y mutat ion.