Using mating-type switching to investigate Smc5/6 function in Schizosaccharomyces pombe

The essential Smc5/6 complex is structurally related to cohesin and condensin. It is required for homologous recombination (HR), rDNA stability and telomere maintenance. In Schizosaccharomyces pombe, two hypomorphic smc6 mutants, smc6-X and smc6-74, haven been shown to be deficient in HR-dependent processing of collapsed replication forks. Collapsed replication forks can generate single-ended DNA double strand breaks (se-DSB) which require HR to restore replication. In this study the requirement for Smc5/6 at a site-specific se-DSB at the mating-type locus and in the mating-type switch process were analysed. In S.pombe mating-type switching occurs over two S phases; in the first S phase replication fork stalling at mat1 leads to an imprint, which is converted to an se-DSB during the next S phase. This initiates the copying of the donor cassette using HR. In the absences of donors the sister chromatid is used for repair. Mating-type switching analysis showed that snc6-74 had a defect in switching dependent on the genotype of the smc6-74 parent. Both smc6 mutants had reduced viability in the absence of donors, consistent with a defect in HR repair of an se-DSB. analysis in an inducible system (Holmes et al., 2005) showed that in response to a se-DSB Rad52 foci appeared with wild type kinetics but the smc6 mutants delayed entry into mitosis for approximately 2hrs, dependent on the DNA damage checkpoint kinase Chk1. In order to test whether this delay facilitated rescue by a converging replication fork a novel inducible converged fork (cf) DSB system was developed. The cf-DSB required HR and the RecQ helicase Rqh1 for repair but did not require Mus81. The converging fork rescued smc6-74 but not smc6-X showing Smc5/6 to be required for repair of both types of replication-associated DSBs.