SUMMARY The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on γH2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50Δ mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a function for cohesin in the maintenance of genome integrity during S phase.

SUMMARYThe Mre11 complex (Mre11, Rad50, Xrs2 in S. cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA binding domain and the Rad50 hook domain which are linked by a long and extended Rad50 coiled coil domain. In this study, rad50 alleles encoding truncations of the coiled coil domain were constructed to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance, meiotic DSB formation, and severely impaired HR, indicating a requirement for long range action. NHEJ, which is likely mediated by the Mre11 complex globular domain was also severely impaired by alteration of the coiled coil and hook domains, providing the first evidence of their influence on this process. These data show that Mre11 complex functions are integrated by the coiled coils of Rad50.

The Mre11 complex (in Saccharomyces cerevisiae: Mre11, Rad50 and Xrs2) influences multiple facets of chromosome break metabolism. A conserved feature of the Mre11 complex is a zinc-coordinating motif in Rad50 called the Rad50 hook. We established a diploid yeast strain, rad50(hook), in which Rad50 is encoded in halves, one from each of the two RAD50 alleles, with the residues constituting the hook deleted. In all respects, rad50(hook) phenocopies complete Rad50 deficiency. Replacing the hook domain with a ligand-inducible FKBP dimerization cassette partially mitigated all phenotypes in a ligand-dependent manner. The data indicate that the Rad50 hook is critical for Mre11 complex-dependent DNA repair, telomere maintenance and meiotic double-strand break formation. Sister chromatid cohesion was unaffected by Rad50 deficiency, suggesting that molecular bridging required for recombinational DNA repair is qualitatively distinct from cohesin-mediated sister chromatid cohesion.

Highlights d The MRX complex stabilizes Nej1 at the break site d Nej1 inhibits Dna2 recruitment and HR d Nej1 and the coiled-coil region of Rad50 are important for tethering broken DNA ends d Defects in 5 0 resection and end-tethering lead to large chromosome deletions at the DSB