Replicated chromosomes are held together by the chromosomal cohesin complex from the time of their synthesis in S phase onward. This requires the replication fork-associated acetyl transferase Eco1, but Eco1's mechanism of action is not known. We identified spontaneous suppressors of the thermosensitive eco1-1 allele in budding yeast. An acetylation-mimicking mutation of a conserved lysine in cohesin's Smc3 subunit makes Eco1 dispensable for cell growth, and we show that Smc3 is acetylated in an Eco1-dependent manner during DNA replication to promote sister chromatid cohesion. A second set of eco1-1 suppressors inactivate the budding yeast ortholog of the cohesin destabilizer Wapl. Our results indicate that Eco1 modifies cohesin to stabilize sister chromatid cohesion in parallel with a cohesion establishment reaction that is in principle Eco1-independent.

After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.

We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring. After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion. In anaphase, Scc1 cleavage opens the ring to release sister chromatids.