Identification of the anaphase inhibitor Pds1/securin led to a greater understanding of how chromosome cohesion is maintained until anaphase in eukaryotes (47
). However, the viability of pds1
-null yeast and securin-null mammals (23
) indicated that cohesion is maintained in S phase independently of Pds1. Pds1 inhibits the protease Esp1, which cleaves the Mcd1/Scc1 component of cohesin. Maintenance of cohesion in S-phase pds1Δ
cells might be a fortuitous result of the reduced Esp1 activity (8
) and compromised nuclear import of Esp1 (25
) when Pds1 is not present. However, loss of cohesion occurs during S phase in pds1
-null cells under conditions of replication stress. Therefore, Pds1-independent mechanisms are required for cohesion when replication is perturbed.
Here we have described evidence that, in budding yeast, the S-phase cyclins Clb5 and Clb6 are important regulators of cohesion. A cohesion defect in clb5Δ clb6Δ cells was observed cytologically (separation of the newly replicated TRP1 loci) and biochemically (reduced abundance of Mcd1/Scc1-Myc at TRP1 and LYS4). Under conditions of replication stress (in HU), either a lack of Pds1 or a lack of Clb5 and Clb6 results in a cohesion defect. Thus, neither of these factors is sufficient when replication fork progression is perturbed. When both Pds1 and the cyclins are absent, cells lose cohesion in S phase even in the absence of the replication stress induced by HU. The consequences are aneuploidy and cell death. We also find that Cdc28-dependent phosphorylation of Pds1 is not required for cohesion in S phase. These data indicate that the function of Clb5 and Clb6 in promoting cohesion does not depend on Pds1 but rather constitutes a distinct mechanism.
Interestingly, we did not observe premature loss of cohesion at the TRP1
locus in clb5Δ clb6Δ
cells treated with HU and nocodazole. We also failed to observe premature loss of cohesion at the LYS4
locus and near the telomere of chromosome IV in clb5Δ clb6Δ
cells treated with HU alone. These experiments raised the possibility that prolonged spindle forces during S phase might pull the centromeric regions further apart, giving rise to the observed separation of the TRP1
loci. Counter to this possibility, in clb5Δ clb6Δ
mutants treated with HU, the mitotic spindle did not elongate until after S phase was completed, indicating that anaphase spindle forces are not prematurely initiated. More importantly, we observed comparably reduced abundances of chromosomal Mcd1/Scc1-Myc at TRP1
in clb5Δ clb6Δ
cells treated with HU. Spindle forces are therefore not required for the cohesin deficiency in clb5Δ clb6Δ
cells because LYS4
is not subject to tension during S phase. Presumably, the lack of TRP1
separation in clb5Δ clb6Δ
cells treated with nocodazole and HU demonstrates that this cytological assay requires spindle force in order for the cohesion defect to be observed. The reduced abundance of Mcd1/Scc1-Myc on chromosomes in S-phase clb5Δ clb6Δ
cells is intriguing given a recent study which demonstrated that reduced levels of cohesin on replicating DNA slow down the replication forks (19
). This suggests that the prolonged DNA replication observed in clb5Δ clb6Δ
mutants might be a consequence of reduced chromosomal cohesin loading.
We also find that loss of cohesion does not occur in clb5Δ clb6Δ esp1
cells. However, we did not observe premature Mcd1/Scc1 cleavage in clb5Δ clb6Δ
cells, indicating that the cohesion defect is not due to premature Esp1 activation. Indeed, the Mcd1/Scc1 cleavage products were less abundant in clb5Δ clb6Δ
cells compared to wild-type cells. Mcd1/Scc1 cleavage occurs preferentially on the chromatin-associated pool of cohesin complexes (22
). The data are therefore consistent with our observation that Mcd1/Scc1 is less abundant on chromatin during S phase in clb5Δ clb6Δ
cells than in wild-type cells. Together the experiments suggest that the cohesin defect in clb5Δ clb6Δ
cells, and the reduced level of cleavage products, is due to a cohesin loading defect in early S phase. The cytological assay, observation of TRP1
locus separation, indicated a cohesion defect in the latter half of S phase. Since loss of cohesion required Esp1 and spindle tension, a model to explain these data is that the low level of chromosomal Mcd1/Scc1-Myc in clb5Δ clb6Δ
cells is sufficient for cohesion until later in S phase when Esp1 activity is increased and spindle forces contribute to pulling the locus apart. However, we cannot rule out the possibility that S-phase cyclins also act directly on Esp1, given that we were able to coprecipitate Esp1 in complexes with Cdc28 and Cks1.
In summary, we describe evidence that specific yeast S-phase cyclins promote loading of cohesin complexes onto chromatin in early S phase (D). In an unperturbed S phase, this mechanism acts in parallel with Pds1 to ensure cohesion is sufficient to hold sister chromatids together. When replication fork progression is stressed, both efficient cohesin loading (Clb5 and Clb6 function) and protection (Pds1 function) are required for cohesion.