Mutants Defective in Rad1-Rad10-Slx4 Exhibit a Unique Pattern of Viability During Mating-Type Switching in Saccharomyces cerevisiae

Abstract

Efficient repair of DNA double-strand breaks (DSBs) requires the coordination of checkpoint signaling and enzymatic repair functions. To study these processes during gene conversion at a single chromosomal break, we monitored mating-type switching in Saccharomyces cerevisiae strains defective in the Rad1-Rad10-Slx4 complex. Rad1-Rad10 is a structure-specific endonuclease that removes 39 nonhomologous singlestranded ends that are generated during many recombination events. Slx4 is a known target of the DNA damage response that forms a complex with Rad1-Rad10 and is critical for 39-end processing during repair of DSBs by single-strand annealing. We found that mutants lacking an intact Rad1-Rad10-Slx4 complex displayed RAD9-and MAD2-dependent cell cycle delays and decreased viability during mating-type switching. In particular, these mutants exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Furthermore, we observed that mutations in post-replicative lesion bypass factors (mms2D, mph1D) resulted in decreased viability during mating-type switching and conferred shorter cell cycle delays in rad1D mutants. We conclude that Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 39 nonhomologous tail and propose that the rad1D and slx4D mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair.

Real-time analysis of double-strand DNA break repair by homologous recombination

“…We note that NH-tail clipping must be able to occur at the point of MAT-HML synapsis because MAT switching is still reasonably efficient when both ends of the DSB contain terminal NH sequences (39,50). This is further supported by the observation that MAT switching can give rise to reciprocal COs (28) thought to occur through a dHJ intermediate, whose formation would require NH-Ya tail clipping at the point of MAT-HML synapsis.…”

“…This process requires Rad1-Rad10, Msh2-Msh3, Slx4, Saw1, and other components of a complex that can remove 3′-ended NH-tails in SSA and in MAT switching (39)(40)(41)(42)(43). To measure NH-tail clipping kinetics during MAT switching, we designed two PCR primers to anneal on either side of the MAT-X-Ya junction (Fig.…”

Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

“…Slx4 also interacts with the Rad1-Rad10 complex, a member of the yeast XPF/MUS81 structure-specific nuclease family required for NER and ICL repair (Flott et al 2007;Ciccia et al 2008). The Slx4-Rad1-Rad10 complex is required for DSB repair during single-strand annealing (SSA) (Li et al 2008;Lyndaker et al 2008). Taken together, Slx4 coordinates multiple DNA repair pathways and recombination events.…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…One strand is of course removed by 59 to 39 resection, but the second one is clipped off-apparently only after the elongated first strand anneals in MAT-X region-by the Rad1-Rad10 endonuclease Lyndaker et al 2008). In other HO-induced events (and probably in MAT switching) removal of the nonhomologous tail also requires a number of other proteins, including Msh2-Msh3, Saw1, and Slx4 (Colaiacovo et al 1999;.…”