Pds5 regulates sister chromatid cohesion by controlling cohesin ATPase activity through the Eco1–Smc3 acetylation pathway

Cohesin is a highly conserved protein complex essential for sister chromatid cohesion (SCC) and proper chromosome segregation during cell division. Pds5, a critical cohesin subunit, is essential for the function and stability of the cohesin complex on chromatin. However, the precise molecular mechanisms underlying Pds5’s essential role remain poorly understood. To elucidate Pds5 function, we employed a genetic bypass suppressor screen to identify genomic mutations that restored viability in the otherwise lethal pds5∆ elg1∆ double mutant strain. Our screen identified three-point mutations in the Smc3 cohesin subunit that rescued the inviability, SCC defects, and reduced Mcd1/Scc1 protein levels associated with Pds5 loss. Remarkably, these smc3 suppressor mutants also rescued the inviability and cohesion defects of the smc3-RR mutant (unable to undergo acetylation by Eco1) and suppressed the temperature sensitivity of an eco1-203 ts allele. Notably, one suppressor mutation, smc3-G1128D, resides within the highly conserved ABC-signature (or C-motif) that is critical for cohesin ATPase activity. Using molecular dynamics simulations and ATPase assays with purified cohesin complexes, we demonstrated that the smc3-G1128D mutant significantly reduces cohesin’s ATPase activity. The bypass smc3 mutants suppress the pds5∆ phenotype by bypassing the requirement for Eco1-dependent Smc3 acetylation in cohesion establishment and cell viability. Our findings reveal that PDS5 functions in a pathway that is epistatic to the Eco1-dependent Smc3 acetylation pathway during cohesion establishment, where Pds5 is strictly required to promote Eco1-dependent Smc3 acetylation and inhibit cohesin’s ATPase activity. Following cohesion establishment, Pds5 ensures cohesion maintenance by safeguarding cohesin complex integrity. This work reveals new molecular insights into Pds5-mediated cohesin regulation and establishes the critical importance of controlled cohesin ATPase activity for proper SCC maintenance.