Reviewer #4 (Public Review):
Higashi et al. provide a new "Brownian ratchet" model for DNA loop extrusion mechanism by cohesin, a member of SMC protein family complexes. Based on previous works on crystal structures, cryo-EM structures, and DNA-protein crosslinking experiments, they shed light on two HEAT-repeat DNA binding modules on cohesin - Scc2-head and Scc3-hinge - and their relationships. They hypothesized that the association between Scc2-head and Scc3-hinge modules were dissociated and Scc2-head released DNA upon ATP hydrolysis, driving DNA slipping. By performing FRET experiments, they found that Scc2 and hinge modules indeed come close only in ATP-bound "Gripping" state, while hinge and Scc3 are always close to each other. Therefore, they suggest that, for DNA loop extrusion model, 1) upon ATP binding to the head domains, both Scc2-head and Scc3-hinge modules grip DNA, 2) when ATPs are hydrolyzed, Scc2-head module releases DNA so that DNA-associating Scc3-hinge module pulls DNA depending on stochastic Brownian motion of Scc3-hinge module, then 3) both Scc2-head and Scc3-hinge modules release DNA and go back to the state 1). This "Brownian ratchet" model also provides an explanation of how cohesin entraps DNA by opening the gate between Smc3 and Scc1, which also nicely explains the known facts regarding Scc1 cleavage-dependent DNA release and in vitro behaviors of single cohesin molecules that topologically bound to DNA. In addition, by performing computational modeling, they showed that the Brownian ratchet model well fits all previously reported in vitro loop extrusion assays by cohesin and condensin, making their model rigid and reliable.
Their model is mostly well supported by data, but several detailed points need to be explained or clarified.
1) In Figure 2C FRET experiments, proximity of Scc3-C and Scc2-N does not seem to be drastically increased in Gripping state compared to the case of hinge and Scc2-N. This could be because the FRET pairs (Scc3-C and Scc2-N) are still far. If the authors could label internal part in Scc3, this could solve the problem. In addition, if Scc3-C and Scc2-N are always close to each other irrespective of Gripping state, the authors should consider this fact in their modeling.
2) Major differences between topological loading and loop extrusion is kleisin-gate opening and head gate passage. Even if kleisin-gate wouldn't be opened, DNA should be released after head opening like in the topological loading. In case it happens, DNA and Scc1 would be tangled and it seems to be difficult to come back to next gripping state again. It would be helpful to add the explanation of why such tangling DNAs do not have to be considered in the model.
3) In the manuscript line 338, the authors mention "After DNA dissociation from the Scc3-hinge module, there is a time without tight contact between the cohesin ring and the DNA loop." However, both in Figure 3B and 4F, it seems that head-Scc2 always associates with DNA. This could be discrepancy. The authors should clarify the point if certain free time without any contact to DNA is assumed in the modeling.
4) Generally, initial DNA bending is the most challenging part in loop extrusion models. Especially in Figure 3B-a, such a bent DNA seems to be impossible if we consider the persistence length of DNA is 50 nm. The authors should discuss how DNA loop extrusion could be initiated.