Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: A role for SAE2/COM1

Errors associated with the repair of DNA double-strand breaks (DSBs) includ e point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NMEJ). We previously de monstrated that DNA synthesis is similar to 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants t hat affect tile fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specif ic DSB within the repeat yields TRP1 recombinants. Errors in the repair pro cess can be detected by the production of canavanine-resistant (can1) mutan ts among the TRP1 recombinants. In wild-type cells the recombinational repa ir process is efficient and fairly accurate. Errors resulting in can1 mutat ions occur in <1% of tile TRP1 recombinants and most appear to be point mut ations. We isolated several mutant strains with altered fidelity of recombi nation. Here we characterize one of these mutants that revealed an similar to 10-fold elevation in tile frequency of can1 mutants among TRP1 recombina nts. The gene was cloned by complementation of a coincident sporulation def ect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chr omosome rearrangement that could reflect. break-induced replication (BIR) a nd NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had ph enotypes in this assay that are similar to that of the sae2/com1 Delta stra in. Our data suggest that Sae2p/Com1p plays a role in ensuring that both en ds of a DSB participate in a recombination event, thus avoiding BIR, possib ly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.