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) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect 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 chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.
Rattray
A J
AJ
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.
McGill
C B
CB
Shafer
B K
BK
Strathern
J N
JN
D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004720:Endonucleases; D005656:Fungal Proteins / Q000235:genetics*; D006238:Haploidy; D008938:Mitosis / Q000235:genetics*; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; C107849:SAE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004720:Endonucleases