Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a back-up function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1 intermediates are not channeled exclusively to non-crossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation in the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells.

The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited ,2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.

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 rad51⌬ 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 pol32⌬ and pol3-ct mutants, defective for components of the DNA polymerase ␦ (Pol ␦) complex. The half crossovers formed in Pol ␦ 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 rad51⌬ mutants, the Pol ␦ 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 ␦ complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.DNA double-strand breaks (DSBs) are potentially lethal lesions that can occur spontaneously during normal cell metabolism, by treatment of cells with DNA-damaging agents, or during programmed recombination processes (54). There are two major pathways to repair DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves the religation of the two ends of the broken chromosome and can occur with high fidelity or be accompanied by a gain or loss of nucleotides at the junction (9). Repair of twoended DSBs by HR generally occurs by gene conversion resulting from a transfer of information from the intact donor duplex to the broken chromosome (Fig. 1). HR occurs preferentially during S and G 2 when a sister chromatid is available to template repair (2,19,22). Sister-chromatid recombination events are genetically silent, whereas gene conversion between nonsister chromatids associated with an exchange of flanking markers can result in extensive loss of heterozygosity (LOH) or chromosome rearrangements (3, 21). One-ended DSBs that arise by replication fork collapse or by erosion of uncapped telomeres are thought to repair by strand invasion into homologous duplex DNA followed by replication to the end of the chromosome, a process referred to as break-induced replication (BIR) (35). BIR appears to be suppressed at two-ended breaks, presumably because it can lead to extensive LOH if it occurs between homologues or to chromosome translocations when strand invasion initiates within dispersed repe...

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

Holliday junctions can be formed during homology-dependent repair of DNA double-strand breaks and their resolution is essential for chromosome segregation and generation of crossover products. The Mus81–Mms4 and Yen1 nucleases are required for mitotic crossovers between chromosome homologs in Saccharomyces cerevisiae; however, crossovers between dispersed repeats are still detected in their absence. Here we show the Rad1–Rad10 nuclease promotes formation of crossover and noncrossover recombinants between ectopic sequences. Crossover products were not recovered from the mus81Δ rad1Δ yen1Δ triple mutant indicating that all three nucleases participate in processing recombination intermediates that form between dispersed repeats. We suggest a novel mechanism for crossovers that involves Rad1–Rad10 clipping and resolution of a single Holliday junction-containing intermediate by Mus81–Mms4 or Yen1 cleavage, or by replication. Consistent with the model, we show the accumulation of Rad1 dependent joint molecules in the mus81Δ yen1Δ mutant.