The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

Abstract

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

“…In addition, there is direct evidence that each sister chromatid is individually decorated by cohesins and that Mcd1 can bridge different Smc1,3 heterodimers (4, 6, 31, 59, 60), providing support of an early model that cohesion is mediated through cohesin-cohesin interactions (61). Intriguingly, Pds5 (and Nse5 for Smc5,6 complexes) bind both the head and hinge domains (62,63), in support of findings that cohesins fold over to promote head-hinge interactions (64, 65)-further challenging a simplistic ring model. Our findings regarding PCNAdependent rescue further support a higher-order cohesin assembly model in that mutant pds5-1 protein becomes resistant to temperature shift inactivation during mitosis.…”

The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker

“…Smc5/6K115E also displayed reduced DNA binding as compared to a wild-type complex in a filter binding assay (compare Figure 6D with Figure 4A). In addition, analysis of established chromosomal binding sites for Smc5/6 by chromatin immunoprecipitation (ChIP) and qPCR revealed that Smc5/6K115E displayed reduced chromosome binding in vivo ( Figure 6E) (Jeppsson et al, 2014). The Smc5/6K115E complex isolated from top2-4 cells was also found to be less efficient than the wild-type complex in stimulating TOP2a-dependent catenation ( Figure 6F).…”

Observation of DNA intertwining along authentic budding yeast chromosomes

“…If cohesin is required to retain sister chromatid intertwining, do intertwines accumulate at cohesin-binding sites? The Smc5/Smc6 complex, implicated in marking intertwines, is also found at cohesin-binding sites (Jeppsson et al 2014). To address this, we excised two comparable replication termination regions on chromosomes III (TER301) and VI (TER603).…”

“…During mitosis, the last remaining catenanes are removed in a reaction that is promoted by condensin and by physical sister chromatid separation by the mitotic spindle (Koshland and Hartwell 1987;Baxter et al 2011;Farcas et al 2011;Charbin et al 2014). The levels of the Smc5-Smc6 complex increase along chromosomes when intertwines accumulate following topo II inactivation, which led to the proposal that the Smc5-Smc6 complex recognizes sites of intertwining (Jeppsson et al 2014). These previous studies did not address where along authentic chromosomes intertwines arise and persist or when during S phase they are generated.…”