Abstract
In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are 'clamped' in a sub-compartment created by Scc2's association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.
Highlights
Protein complexes containing SMC and kleisin subunits organise the spatial arrangement, or topology, of DNAs in most if not all living organisms (Nasmyth, 2001; Yatskevich et al, 2019)
Our observations reveal key insights into the biochemical activities of Scc2 and Scc3 and suggest that the recurrent clamping of DNAs by Scc2NIPBL/Mis4 and engaged heads resulting in E-S/E-K entrapment, followed by their subsequent release, may be an integral aspect of cohesin’s ability to load onto and translocate along DNA
In vivo studies have shown that cohesin entraps circular minichromosomes within its SMC-kleisin ring (S-K) ring (Gligoris et al, 2014; Srinivasan et al, 2018)
Summary
Protein complexes containing SMC and kleisin subunits organise the spatial arrangement, or topology, of DNAs in most if not all living organisms (Nasmyth, 2001; Yatskevich et al, 2019). Though the mechanism by which cohesin extrudes loops remains mysterious, there is a clear and simple hypothesis as to how cohesin holds sister DNAs together, namely by entrapping them both inside the S-K ring created through the binding of a kleisin subunit to the ATPase heads of an Smc1/ Smc heterodimer (Gruber et al, 2003; Haering et al, 2002) This model explains the key observation that cleavage of cohesin’s kleisin subunit by separase, or any other site-specific protease, is sufficient to trigger sister chromatid disjunction at anaphase (Oliveira et al, 2010; Uhlmann et al, 2000). Our observations reveal key insights into the biochemical activities of Scc and Scc and suggest that the recurrent clamping of DNAs by Scc2NIPBL/Mis and engaged heads resulting in E-S/E-K entrapment, followed by their subsequent release, may be an integral aspect of cohesin’s ability to load onto and translocate along DNA
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