Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

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.

Condensin complexes: understanding loop extrusion one conformational change at a time

“…This bend, referred to as the 'elbow', has also been observed in EM analysis and crosslinking data of S. cerevisiae and S. pombe cohesin and E. coli MukBEF SMC-Kleisin, and results in the hinge bending to contact the SMC arms near the ATPase domains [40,60]. In cohesin, crosslinking and FRET based studies support the hypothesis that the hinge may fold to contact S. pombe Pds5, Psc3 or Mis4 [49,61,62] and cryo-EM structures of H. sapiens, S. cerevisiae and S. pombe suggest that the hinge folds to contact the HAWKs [39][40][41]. Folding of the SMC arms is observed in recent AFM data of S. cerevisiae condensin, showing that the hinge can fold towards the ATPase domains, however, does so with the SMC arms open, transitioning from an open O to a B shaped conformation [63].…”

“…Current models of S. cerevisiae cohesin and B. subtilis Smc-ScpA [69,70] suggest SMC complexes have of two distinct entrapment compartments, referred to as the S compartment (as it is formed by the SMCs), and the K compartment (formed by Kleisin), which can fuse to create a larger S/K ring ( Figure 3A). The investigation into K and S compartments in S. cerevisiae cohesin suggest Scc2 promotes ATP independent DNA entry into both the S and K compartments, but not the S/K ring, while Scc3 and ATP hydrolysis is required for DNA entry into the K-compartment [41]. Cryo-EM work in this study, and by others on S. pombe and H. sapiens cohesin, show that DNA is gripped by Scc2 on one side and trapped by engaged ATPase heads on the other [39][40][41], hence, DNA could gain entry to the S and K compartments by DNA binding to Scc2, before the ATPase heads close around it.…”

“…The investigation into K and S compartments in S. cerevisiae cohesin suggest Scc2 promotes ATP independent DNA entry into both the S and K compartments, but not the S/K ring, while Scc3 and ATP hydrolysis is required for DNA entry into the K-compartment [41]. Cryo-EM work in this study, and by others on S. pombe and H. sapiens cohesin, show that DNA is gripped by Scc2 on one side and trapped by engaged ATPase heads on the other [39][40][41], hence, DNA could gain entry to the S and K compartments by DNA binding to Scc2, before the ATPase heads close around it. Overlay of the S. cerevisiae Apo condensin structures with NIPBL/SMC1/DNA from H. sapiens cohesin structure [39], suggest condensin could bind DNA in a similar manner (Figure 3B,C).…”

“…In single-molecule experiments, while condensin remains bound to DNA after ATP is washed out [17], loss of ATP or NIPBL/MAU2 results in human cohesin loop release [28], suggesting that cohesin is less stably tethered to DNA. This could be explained by the discovery of the 'gripping state' where Scc2/NIPBL within a cohesin complex firmly grips DNA in the presence of ATP [39][40][41]. In the case of human cohesin, lower DNA association stability might be compensated for by additional DNA binding factors, such as CTCF, which binds directly at the Scc3/Scc1 interface [42].…”

“…Hence, folding of the SMC hinge towards the globular region ( Figure 1G) is a conserved feature in SMC complexes. As the SMC hinge domains have been found to bind DNA [64][65][66][67][68] and S. cerevisiae condensin and cohesin have been observed to interact with DNA via both their hinge and globular domains [41,63], folding is likely to have a key role in the loop extrusion mechanism.…”