Aberrant double-strand break repair resulting in half crossovers in mutants defective for Rad51 or the DNA polymerase delta complex.
Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Delta mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Delta and pol3-ct mutants, defective for components of the DNA polymerase delta (Pol delta) complex. The half crossovers formed in Pol delta complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Delta mutants, the Pol delta complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol delta complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.
Smith
Catherine E
CE
Columbia University College of Physicians & Surgeons, Department of Microbiology, New York, NY 10032-2704, USA.
Lam
Alicia F
AF
Symington
Lorraine S
LS
D019457:Chromosome Breakage; D015825:Chromosomes, Fungal / Q000235:genetics / Q000502:physiology; D053903:DNA Breaks, Double-Stranded; D004258:DNA Polymerase III / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D009154:Mutation; D051135:Rad51 Recombinase / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology
D029701:Saccharomyces cerevisiae Proteins; D051135:Rad51 Recombinase; D004258:DNA Polymerase III