Assays of cohesin–NIPBL complexes bearing mutations in individual subunits identify DNA-binding sites in the hinge, ATPase heads and NIPBL that are required for loop extrusion, and show that NIPBL and SMC3 head sites form a ‘DNA clamp’ that stimulates cohesin’s ATPase activity. Imaging of cohesin by high-speed atomic force microscopy (HS-AFM) reveals that, when ATP is present, complexes alternate between ring-shaped, rod-shaped and bent conformations that modulate the interactions between the head domains and the distance between the SMC3 head and the hinge in a mutually exclusive fashion. Thus, the DNA-binding sites within these domains move relative to each other, and DNA could be translocated over long distances by these movements. Single-molecule fluorescence resonance energy transfer (smFRET) assays further show that the ATP-dependent head domain movements and the ATP-independent coiled-coil alignment and hinge bending are mutually exclusive and function at distinct DNA translocation steps driven by cycles of ATP binding and hydrolysis. ATP binding leads to head engagement, coiled-coil opening and straightening, whereas subsequent ATP hydrolysis causes head disengagement that permits coiled-coil alignment and hinge bending. The movements between the hinge and heads suggests that DNA is transferred between these sites during translocation, coupling cohesin’s DNA-binding and ATPase activities. Indeed, the authors find that the DNA clamp formed by the SMC3 ATPase head and NIPBL is assembled and disassembled during each ATP binding–hydrolysis cycle. The ‘swing and clamp’ model predicts that NIPBL associates with the hinge and is ‘delivered’ to the SMC3 head by hinge bending (the ‘swing’). DNA translocation occurs during this step, with the DNA transferred from the hinge to the head domains. Upon ATP binding, NIPBL and the SMC3 head form a DNA-binding clamp that promotes interactions between the head domains, and the coiled coils straighten. Head engagement triggers ATP hydrolysis, leading to head disengagement and clamp release to reset cohesin for another DNA translocation cycle.
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