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  1. 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.

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  2. Reviewer #3 (Public Review):

    Bifurcation between topological loading and loop extrusion is determined by DNA passing through the N-gate. For loop extrusion to occur processively, this decision needs to be made only once at the beginning. However, the authors also argue that Scc2 dissociation between rounds of ATPase cycles is required for symmetric loop extrusion. In combination, the model requires that N-gate opening is allowed only at the very beginning and cannot occur during loop extrusion, even when the cohesion loader is released. The authors should state whether this interpretation is correct and feasible given the structural data.

    Loop extrusion has never been observed using yeast cohesin. It will be important to learn how the authors reconcile their model and the lack of experimental demonstration of loop extrusion in a reconstituted system.

    The discrepancy in speed and the measured ATPase rate is not discussed. In vitro, loop extrusion rates are about 1000 bp per second and in vivo measurements of gamma-H2AX spreading from a double strand break, ~150kbp per min according to PMID: 32527834, which was proposed to be caused by loop extrusion (PMID: 33597753), also matches that in vitro rate. But the authors model accounts for only about 100 bp extrusion per ATPase cycle whereas the average ATPase rate is 1 per second. They do mention that the model requires 9 ATP hydrolyzed per second but do not make an attempt to explain the discrepancy.

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  3. Reviewer #2 (Public Review):

    How the genome chromatin fiber is folded into loops and topologically associating domains (TADs) remains unclear. A recent attractive model is that these genomic structures are formed by a loop extrusion process mediated by cohesin. While the Uhlmann group has proposed an alternative mechanism, the diffusion capture model, to make loops (Cheng et al., 2015; Gerguri et al., 2021), in this paper, Higashi et al. proposed a structure-based model providing mechanistic insight into the reported loop extrusion activity of cohesin. For its topological DNA binding, cohesin inserts DNA into the cohesin ring by sequential passage through a kleisin N-gate and an ATPase head gate. Hisgashi et al. suggested that the gripping state in which DNA has not passed the kleisin N-gate might facilitate the loop extrusion activity reported. This paper is very intriguing, and informative to the chromatin/chromosome field. My specific comments are the following:

    1. Since this paper is primarily based on the detailed structural information on cohesin loading onto DNA, which the Uhlmann group published in Mol Cell (2020), it might be hard for general readers to follow the whole story in this paper. For better understanding, the authors should provide readers with Supplemental Fig. corresponding to the Graphical abstract and Figs. 6E/7G in the Mol Cell paper, and adequately explain it first. Structural models such as Fig. 1 are accurate but might be difficult to capture cohesin's dynamic behavior with DNA.

    2. Although this paper is very intriguing, it looks like a review paper, and the authors' message is not so clear. Given that the Uhlmann group has proposed an alternative mechanism to make loops, I wonder whether the main message might be that the loop extrusion, like reported in vitro, is unlikely to occur in vivo. If so, the authors should clearly state the point and shorten the Discussion part to enhance the paper's impact.

    3. Page 24. The critical issue of the loop extrusion mechanism proposed is "not opening" of kleisin N-gate. The authors discussed that the low salt condition in vitro could be a reason: " For instance, electrostatic interactions contribute to keeping the kleisin N-gate closed and these are augmented in a low salt buffer." However, I assume that the condition also helps the topological loading, and this explanation is not so convincing.

    4. While I agree with the authors' loop extrusion mechanism, there are other models to explain cohesin loading onto DNA (e.g., Shi et al., 2020; Collier et al.). They might want to discuss its compatibility with them.

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  4. Reviewer #1 (Public Review):

    Higashi et al. present a molecular mechanism of how the cohesin complex, a supramolecular assembly of several proteins, can topologically embrace DNA and actively extrude DNA into loops. The loop-extruding activity of cohesin and of related condensin complexes have been proposed to represent a cornerstone of genome organization. While recent in vitro studies demonstrate that cohesin and condensin complexes are capable of extruding loops, the molecular mechanisms driving loop extrusion, i.e. how ATP energy is utilized, and what underlies the processivity of the loop extrusion, remains enigmatic. The cohesin complex consist of two long flexible protein "arms" connected at the 'hinge' ends. The other, 'head' ends are linked by the kleisin protein and also can dimerize to form an ATP-binding chamber. Defining how transitions in the cohesin complex structure and its ATPase activity underlies known cohesin functions has been the object of numerous studies for over two decades. Here, the authors build upon these studies.

    The authors start by analyzing available structural data for cohesin domains and associated loading factors. First, by combining the structure of the cohesin-head-domain complex engaged with DNA in ATP-bound state and the corresponding free crystal structures, they show that the 'head module' in the ATP-bound state can tightly wrap around DNA, and upon ATP hydrolysis the DNA-embracing cavity will dilate. In other words, the complex transitions from a 'DNA-gripping state' into a 'DNA-slipping' state after ATP is hydrolyzed. Next, they show that the other DNA-binding module, the 'hinge module', does not change its interaction with the DNA after ATP hydrolysis. The authors also conclude that ATP hydrolysis weakens the interaction between the head and hinge modules, suggesting that the cohesin ring alternates between folded (with head and hinge closed) and unfolded ('free' hinge) states. The authors next carried out FRET experiments to provide experimental evidence for the predicted change in spatial arrangement between the head and hinge modules. Based on this structural analysis, they propose that whether DNA is passed (or not) through the 'kleisin gate' before binding to the head module (into the gripping state) determines if the DNA will be released inside the cohesin ring (i.e. 'topological entry') or if the DNA will remain loosely associated with the head module (i.e. 'loop extrusion') upon ATP hydrolysis. In the latter case, repetitive simultaneous binding of DNA to the head and hinge modules in a folded state followed by relaxation of the cohesin ring while DNA remains bound to the hinge module, may result in a overall 'inward' directed motion of the DNA thread relative to the head domain, i.e. loop extrusion. Stochastic simulations of a coarse-grain model further support that such a model can give rise to loop extrusion.

    The real strength of the paper is in its combination of several pieces of structural and biophysical data that results in a compelling mechanism for cohesin function. The outcome is a united model for cohesin's two characteristic activities - topological engagement of the DNA and DNA loop extrusion. Importantly, the authors explore the role of ATP hydrolysis in driving conformational changes, and, thus, the translocation of DNA, as well as the role of the DNA binding kinetics. The authors go on to relate these findings to the consequences for cohesin function inside cells, where it must content with chromatized substrates. For example, they suggest that while a single nucleosome probably can be bypassed by the cohesin complex, an array of the nucleosome may present a significant hindrance.

    Given its interdisciplinary nature and important conclusions, I believe that this paper will be of broad interest to scientists across disciplines and will influence and stimulate future consideration of how cohesin contributions to the spatiotemporal organization of chromatin.

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  5. Evaluation Summary:

    This work combines experiments and simulations together with previously reported biophysical and structural observations to develop a structure-based model that provides mechanistic insight into the two functions of cohesin: cohesion and loop extrusion. This intriguing and informative manuscript will be of broad interest to those working in the fields of chromatin structure, chromosome biology and molecular machines. While the data and analysis support the authors' conclusions, the presentation of the work can be improved for clarity.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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