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  1. Evaluation Summary:

    This paper addresses the mechanism of entrapment of DNA in the cohesin SMC complex. Through a series of biochemical studies, the paper convincingly demonstrates that DNA enters cohesin rings through the hinge and SMC3/SCC1 interfaces. How such entrapment is regulated is important for different biological activities including sister chromatid cohesion and the formation of DNA loops. The paper will be of interest to researchers in SMC biology, DNA recombination and 3D genome organization.

    (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. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The cohesin ring model postulates that DNA entry and exit must occur through one of the ring's three interfaces thus leading to entrapment. The authors previously tested this model in vitro by engineering disulfide crosslinkers into the different interfaces. Here the authors further test this model by generating cohesin complexes in which the different interfaces can be covalently closed. Using these variants, the authors show that entrapment of DNA can occur through the hinge and SMC3/SCC1 interfaces. Removal of SCC2 and/or SCC3 shows that these regulatory proteins contribute to DNA entrapment through these interfaces, respectively. Sealing of the hinge interface does not prevent entrapment indicating that transport occurs through the passage between the SMC1 and SMC3 ATPase heads. Their data are consistent with the model that DNA entrapment through the SMC and kleisin compartments can lead to initial entrapment. Opening of the hinge may be required for the establishment of cohesion while an opening of the SMC3/SCC1 interface may be required for release. Overall, this information advances our understanding of the molecular basis of DNA entrapment in the cohesin complex.

  3. Reviewer #2 (Public Review):

    The ATPase machine cohesin topologically entraps sister chromatids to build cohesion. It also non-topologically clamps DNA to extrude DNA loops. How cohesin interacts with DNA to execute these two major functions is not understood. In particular, the DNA entry gate of cohesin for the topological entrapment of DNA is debated. Both the hinge and the Smc3-Scc1N interface have been suggested as entry gates. Using covalent fusion proteins and chemical crosslinking, the current study clearly demonstrates that DNA can enter the Smc-kleisin ring of the budding yeast cohesin through either the hinge or the Smc3-Scc1N interface. Strikingly, DNA passage through the hinge requires the cohesin loader Scc2 whereas DNA entry through the Smc3-Scc1N interface does not. As Scc2 is required for topological entrapment of DNA by cohesin in vivo, these findings establish the hinge as the functionally relevant DNA entry gate. The Smc3-Scc1N interface serves as the DNA exit gate but can act as an entry gate under certain conditions in vitro.

    The authors then introduced cross-linkable cysteine pairs based on cryo-EM structures of the clamped state of cohesin. Their results are consistent with the DNA-binding mode revealed by the cryo-EM structures. More importantly, they show that the formation of this clamped state does not require the opening of any of the three cohesin interfaces. This state likely precedes topological entrapment.

    Their findings significantly advance our understanding of how cohesin entraps DNA, defines the functional DNA entry gate of cohesin, and provides biochemical confirmation of the clamped state of cohesin. For these reasons, the study should be of great interest to scientists in the fields of chromosome biology and Smc proteins.