DNA entry into the cohesin ring through the kleisin N-gate

The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion, a prerequisite for faithful chromosome segregation during cell divisions. How DNA enters the cohesin ring remains incompletely understood. Both the interface between Psm3 ^Smc3 and the Rad21 ^Scc1 N-terminus - the N-gate - and the SMC hinge interface - the hinge gate - can open in vitro to entrap DNA. Here, we combine biochemical with in vivo functional assays in the fission yeast S. pombe to probe the DNA entry mechanism. Locking the hinge gate does not interfere with cohesin loader-and ATP hydrolysis-dependent DNA entrapment. Conversely, cohesin with a tightly locked N-gate entraps DNA in vitro but does not load onto chromatin or establish sister chromatid cohesion in vivo . The cohesin ATPase is functionally coupled to N-gate, but not hinge-gate, opening. Our results put forward the N-gate as the primary DNA entry gate into the cohesin ring.