Highlights d 3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate d DNA is trapped between two gates that lead into the cohesin ring d The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state d ATP hydrolysis opens the head gate to complete DNA entry

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively, cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities. ATP and DNA binding promote cohesin conformational changes that guide DNA through a kleisin N-gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin’s heads and hinge, are now juxtaposed. Gripping state disassembly, following ATP hydrolysis, triggers unidirectional hinge module movement, which completes topological DNA entry by directing DNA through the ATPase head gate. If head gate passage fails, hinge module motion creates a Brownian ratchet that, instead, drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian motion has important implications for chromosomal cohesin function.

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring.Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an Nterminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

Our results demonstrate that pre-RC formation regulates chromatin association of XEco2 in Xenopus egg extracts. We propose that this reaction is critical to acetylate cohesin, whose DNA binding is subsequently stabilized by DNA replication.

The biochemical reconstitution of topological DNA binding by budding yeast cohesin yields surprises and opens opportunities to exploit experimental approaches developed in this model organism.