Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection

  1. Martin Scherzer
  2. Fosco Giordano
  3. Maria Solé Ferran
  4. Lena Ström

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Abstract

Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin’s DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae . We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.

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  1. Version published to 10.26508/lsa.202101244
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    Reply to the reviewers

    Response to reviewer comments on:

    “Recruitment of Scc2/4 to double strand breaks depends on γH2A and DNA end resection”, by Martin Scherzer et al

    We would like to thank the editors and reviewers for their time spent, as well as their appreciated and insightful comments on our manuscript. We have now initiated the revision as outlined point by point below. We provide a description of the plan for how to resolve the points of concern still remaining and also list the modifications and improvements already incorporated in the revised and transferred manuscript.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): ____ __In the manuscript entitled "Recruitment of Scc2/4 to double strand breaks depends on yH2A and DNA end resection", Scherzer et al. study the role of Scc2 in DSB repair in yeast. Scc2 is part of the cohesin loader and it is required for cohesin loading in response to DSB. The authors study the chromatin association of Scc2 by ChIP-qPCR and use genetics to identify factors that affect its recruitment. They show that Scc2 is enriched up to 10 kb from the break site, similar to cohesin and identify MRE, TEL1 and yH2A as important factors for Scc2 chromatin binding. Remarkably, MEC1 that has been shown to regulate cohesin under these conditions is dispensable for Scc2 recruitment. While DNA resection is important for Scc2 recruitment, chromatin remodelers don't play a significant role in it despite numerous reports on their effect on cohesin loading during the cell cycle. The manuscript provides new and important information on cohesin regulation in response to DNA damage.

    **Major comments:**

    The experiments are done appropriately and contain the required control. The results are presented clearly and with adequate statistics and support the conclusions. The experiments provide valuable information. However, the low resolution of the experimental setup is limiting, and dynamic information of Scc2 binding is lacking. I would agree with the authors that this kind of information may be beyond their scope. However, the absence of this information reduces the overall impact of the manuscript.

    1. ChIP-seq, of at least some of the key experiments, could provide information on the specific Scc2 binding sites and elucidate whether cohesin is translocated from the loading sites or accumulate in its proximity.

    ChIP -seq would indeed increase the resolution of the Scc2 and Cohesin DSB accumulation, especially beyond 1 kb. However, to gain insight into the dynamics of the binding, numerous timepoints for both strains would have to be analyzed, which we feel would be beyond the possibilities for this study (see also comment under point 4 of this document). For Scc2 we believe that we have shown high enough resolution, determining binding from 0,1 to 30 kb away from the break. We have also provided a time course experiment from 90 minutes up to 6 hours and show that the Scc2 binding is continuously increasing. We have in the revised version of the manuscript added experiments looking at the Cohesin binding in close vicinity of the break – similar to what we previously did for Scc2. With this we confirm the binding pattern of Cohesin previously reported. We have also compared Cohesin binding at 90 and 180 min after break induction, for increased information on the dynamics of its binding at the DSB, and see no change in Cohesin positioning in relation to the DSB site. Rather the general level of binding increases equally over the region, with time (compare Fig 1B and 4A with Fig 1C and Fig S3). This to us indicates that there is no translocation of Cohesin from one loading site to final binding sites. However, to further clarify this issue we plan to include ChIP qPCR experiments on an ATPase deficient mutant of Cohesin, which has been found to be able to be loaded on DNA but not translocated (Hu et al 2010, “ATP Hydrolysis is required for relocating Cohesin from sites occupied by its Scc2/4 loading complex”). These experiments will potentially allow us to explore the possibility that Cohesin is loaded at one (or several) site(s) in the DSB region and then translocated away to the final binding locations with time. The generation of such a strain is ongoing and the results from these experiments will be included in a fully revised version of the manuscript.* *

    1. It has been suggested that Scc2 and Pds5 are mutually exclusive in cohesin complexes. It would be interesting to check in the current experimental setup (ChIP-qPCR) if Pds5 is mimicing Scc2 pattern

    We have generated a strain where Pds5 is FLAG-tagged, and include experiments determining the loading/binding of Pds5 at the break region in the revised version of the manuscript. These show (Fig S1B) that the binding of Pds5 mimics that of Cohesin, indicating that it binds as part of the Cohesin complex. In addition, it is seemingly not affected by the presence of a DSB and therefore most likely not important for the Scc2 or Cohesin loading at the DSB.

    **Minor comments:**

    1. Adding a threshold line to the graphs at fold change= 1 (no enrichment in respect to wild type) will increase their readability.

    We appreciate this suggestion, this has now been added, and is indeed helpful.

    1. Fig. 1A- Add times to the schematic. Modify the text to GAL addition/break induction.

    Thank you for the good suggestion, the figure has now been modified.

    1. Page 9. The authors write: "Cohesin failed to be loaded at the DSB in a mec1Δ background (Fig 3A)". However, the figure shows reduced cohesin binding in mec1delta in respect to the wild type.

    In this graph Cohesin binding in response to break induction is shown. The level of binding in the mec1 deletion mutant is comparable to that of Cohesin in the absence of break induction, See Fig S3 for a newly added experiment showing wt binding of Cohesin at the same timepoint. The text describing Fig 3A on page 9 has also been slightly modified.

    1. Page 10. ".......recruitment to the DSB compared to wild type (Fig 3D)."Should be Fig. 4D.

    Thank you for noticing this mistake, this has now been corrected.

    1. Figure legend 3. "........Protein samples were taken after 3 hours arrest (G2/M, lane 1),....." The benomyl arrest is referred to as G2 arrest in the text but G2/M arrest in the legend. Consistency is needed.

    We agree on the need for consistency and have thus changed to G2/M throughout the manuscript.

    I suggest presenting the suggested model in a figure

    We plan to add an illustrative model figure as Fig 6 in a fully revised version of the manuscript.

    Reviewer #1 (Significance (Required)):

    I am an expert in cohesin biology.

    The Scc2-Scc4 complex has been identified as an essential factor for cohesin loading during the cell cycle (Ciosk et al., 2000). This function has been shown to be essential for cohesin role in response to DNA DSB (Unal et al., 2004, Strom et al., 2004). The interplay between Scc2 and the cohesin has been studied mostly in the context of the cell cycle. It has been shown that Scc2 activates the ATPase activity of cohesin and promotes its translocation from the loading site. Scc2 and Pds5 are mutually exclusive and their switch suppresses cohesin ATPase activity (Hu et al., 2011, Petela et al., 2011). However, the Scc2-cohesin interplay has been poorly studied in the context of DNA repair. The current work adds valuable information on the factors that recruits Scc2 to the break site and identifies end resection as the key event in this process. This information is novel and important and its contribution to the fields of cohesin and DNA repair should not be overlooked. However, ChIP-seq information can increase the overall impact.

    We appreciate the nice verdict. We do agree to some extent on the ChIP seq comment, however based on the discussion under major points 1, we do not see that adding ChIP sequencing experiments to this study will be possible.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Cohesin is a key structural component of chromosomes. Amongst its functions, cohesin plays a critical role in ensuring the accurate repair of double stranded DNA breaks (DSBs). Intuitive as this may seem, a number of fundamental open questions remain. One of these questions is, how does the cohesin loading machinery recognise a DSB? This issue is addressed in the present study. The manuscript begins with a well-written introduction into the fields of DSB repair, as well as cohesin. The research aim is clearly laid out. Experiments follow that sequentially investigate known steps of the DSB repair pathway, asking how these steps intersect with the cohesin loading machinery.

    On the positive side, this is a technically very well conducted study (investigating the cohesin loader has proven tricky in many contexts). The study is systematic and explores the known steps during DSB repair for their impact on cohesin loader recruitment. The authors find a surprising separation of function. The DSB pathway up until H2AX phosphorylation and DNA end resection is required for both cohesin loader recruitment, as well as consequently for cohesin loading. The Mec1 checkpoint kinase, in contrast, is dispensable for cohesin loader recruitment but is required for cohesin loading. This suggests that Mec1 supports cohesin loading at a step beyond that of attracting the cohesin loader. The manuscript thus contains important information that will be of interest to a wide range of researchers in the DNA repair and cohesin fields.

    The limitation of the study lies in the fact that the molecular determinant for cohesin loader recruitment to DSBs remains unknown. H2AX phosphorylation and DNA end resection are shown to be prerequisites, but how do these events form a molecular mark that the cohesin loader recognises? And what is this mark? Equally, how does the Mec1 kinase permit cohesin loading additionally to the cohesin loader?

    We appreciate the positive comments as well as the criticism. We are unfortunately fully aware of the lack of precise knowledge regarding the actual mark made by phosphorylation of H2A, and resection, for recruitment of Scc2. The same is true for the limited understanding of what the exact contribution of Mec1 for Cohesin loading is. We would have liked to execute a screening based approach to find the single determinant – however this has to be performed outside the scope of this study.

    **Specific comments:**

    Figure 1. It would be interesting to overlay the Scc2 prolife around the DSB next with that of Scc1 (obtained previously under similar conditions?), to contrast the loading site with the final cohesin distribution.

    In the revised version of the manuscript, we have looked at the binding of Cohesin close to the break and outwards in the same way as for Scc2, with this experimental system. These binding profiles are not overlapping shown as Fig 1B and 1C. Their different distribution is very clear. This also confirms what been reported previously for Cohesin binding, where the region closest to the break is in principle rather devoid of Cohesin (Fig 1C). This binding pattern is also not changed with increased time for break induction (Fig S3), indicating that there is likely no major translocation of Cohesin from a loading site to the final binding sites around the DSB, at least not during the time frame analyzed, but rather an overall increase in Cohesin binding in the break region. While we cannot exclude translocation completely, we hope that experiments using a Cohesin transition state mutant, deficient in translocation, will address this better.

    Figure 2. Using the same y-axis scale from 1-4 amongst panels A-D could make evaluation of the data easier.

    We agree the comparison is made easier when the scale is the same - this has now been changed within figures.

    Figure 3. Panels A and B contain data that are important to interpret the DNA end resection results shown in Figure S2. Maybe that latter data, which conveys the main conclusion from the figure, could be incorporated within the main figure?

    *This is a good point and we have changed accordingly, now resection experiments in the absence of Scc2 from Fig S2 are shown as Fig 3C. *

    Figure 5. In this figure, the authors begin to investigate possible contributions of candidate cohesin loader receptors, in the form of chromatin remodelling complexes. The Swr1 and INO80 remodellers have an effect on DNA end resection that parallels the effect on Scc2 recruitment, suggesting that their main contribution might be that of facilitating DNA end resection.

    This relationship remains less well documented in the case of Sth1 depletion. Both when using the sth1-3 allele, or degron depletion, the authors observe a relative reduction of cohesin loader recruitment, compared to what they would otherwise expect. However, in both cases a side-by-side analysis of a similarly-treated wild type strain is missing. Whether or not RSC inactivation impacts cohesin loader recruitment therefore remains uncertain.

    *In the revised version of the paper we have included experiments where wild-type cells were grown in the same culturing system as the Sth1 degron strain, included as Figure 5A. The best control would be to use the Sth1 degron strain and not degrade Sth1 as the wt control. However the poor growth of these cells in -Met media with raffinose as the sole carbon source is not compatible with the design of this experiment. *

    For the experiment including the ts allele of Sth1 the wt control was not possible to keep arrested in G2 during the course of the experiment. We agree that a comparison with a wt control would be interesting, however due to not having a proper readout for the impairment of sth1 we decided to omit the data from the ts strain in the manuscript. Based on our results we would conclude that Sth1 inactivation affects Scc2 recruitment due to impaired end resection, deem it unlikely though that this is mediated by direct interaction, as has been shown in S-phase.

    It is also not documented what the corresponding effect of RSC inactivation on DNA end resection might be. Given that previous results suggested that RSC might contribute to cohesin loading at DSBs, the nature of how RSC does this could maybe be clarified before publication.

    In the revised version of the manuscript we are including RPA ChIP data for the Sth1 – degron strain. These show that resection is slightly, albeit significantly, reduced after degradation of Sth1. We believe this to be the explanation for the reduced Scc2 loading in its absence, in line with what is seen in the swr1 and nhp10 deletion mutants.

    Reviewer #2 (Significance (Required)): see above.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    This paper presents data analysing the recruitment of Scc2 to double strand breaks. It makes the interesting observation that its recruitment is Tel1 but not Mec1 dependent, and does not require remodelers (it seems). It does correlate with resection but the mechanism of loading is unclear. I have a few issues on controls and alignment of text with results in this manuscript. Also there is some omission of important recent work and some old studies. But if these points can be resolved it could be published.

    **Major points:**

    1. The cut efficiency under all conditions tested needs to be presented and the CHIP needs to be normalized in every assay to the cut efficiency. This is particularly relevant in the mutants of remodelers as they definitely influence the efficiency of Gal-HO induction. This must be included for every chip result.

    We agree that the Cut efficiency could influence the degree of recruitment due to the strength of the signal from the break for recruitment of the initial DSB response factors that we show are important for recruitment of Scc2. Already in the previous version of the manuscript we therefore show in Fig S3C that the cut efficiency of the chromatin remodelers was comparable to that in WT cells after 3 hours. We have now repeated this type of experiment three times for most strains used in the study and calculated an average cut efficiency for each strain, which is then used for normalization of the ChIPqPCR results. Alternatively, we have used an RT-PCR based method for quantification of the Cut efficiency on the actual ChIP samples when available. The average Cut efficiency is indicated for each strain in the figure legends in the new version of the manuscript. N*ormalization of the ChIP data to the Cut efficiency does in general not change the results or conclusions presented previously, throughout the manuscript. *

    The arp8 delta mutant is clearly polyploid and probably has some suppressor mutation or another problem. They should discard the arp8 results and get a proper and controlled arp8 delta strain (from another lab in europe - there are several with good W303 strains).

    We have repeated the Arp8 transformation in different W303 strains which likewise resulted in polyploidy. Loss of INO80 components have been shown to confer polyploidy in a S288C background, with the loss of Arp8 being an exception. Considering the apparent differences regarding INO80 (the INO80 ATPase subunit is essential in W303 but not in S288C), we deemed it plausible that polyploidization could be a resulting phenotype of an Arp8 deletion in W303. Prompted by the comments put forward here we have now transformed a clean W303 background wild type strain and indeed see no sign of polyploidy. It could be that polyploidization is a consequence of the presence of the GAL:HO in combination with an extra recognition sequence for HO. We are now preparing crosses to answer this question. Depending on the outcome these experiments might be added to a final revision of the manuscript. In this version of the manuscript the arp8delta experiments have been removed.

    1. The text does not accurately reflect the results in several places. For instance .. on page 10 where the result of sgs1 exo1 mutant strain is described, it is said that "Recruitment of Scc2 to the DSB was drastically reduced.... and "consistent with long range resection the effect was less promiment closer to the break.". First, the word "drastic" is not appropriate for a drop of about 50% (on average) and in reality the drop is more significant near the cut (+1kb) than far from the break (+ 10 or 30 kb).... - the data are the opposite of what is stated. and it is not drastic. I do not contest that it correlates with resection, if the HO-cut efficiency is equal in all strains.

    We are sorry for this discrepancy between the results shown and the description of the same in a few cases. We have reworded the results section to reflect the data more accurately. We have also removed the sgs2exo1 deletion mutant data close to the break as we have not investigated all mutants in the region closest to the break and thereby lack a comprehensive comparison.

    The results with INO80 and SWR1 are not really compelling - what is the cut efficiency in these strains. Moreover, the "confusion" in the literature is only because people look at different loci and different conditions. INO80 does affect resection (see Van Attikum et al., 2007; and Cheblal A et al., Molecular Cell 2020) for resection assays in wt and mutant strains. And it is very strange that the Van attikum et al., Cell 2004 (the back to back paper with Morrison et al Cell 2004) is not cited. The data on resection is clear in this early work. But it appears that the arp8 mutant used has other mutations and polyploidization, and should clearly be discarded. Nhp10 impact is a bit controversial but not arp8 with a good strain. The references in general are missing Cheblal A et al., Molecular Cell 2020 for Cohesin recruitment, impact on resection and arp8 impact and ditto. Also missing is Deshpande I et al., molecular Cell 2017 for RPA-Ddc2-Mec1 interactions. These omissions are strange and in fact create confusion in the ms.

    We would like to thank the reviewer for bringing our attention on some very relevant articles published in the field that has now been references as we hope correctly. We have in the revised version of the manuscript also adjusted the ChIP qPCR results to the average efficiency of break induction.

    **Minor points:**

    The english usage needs to be corrected at a few places... and figures are not correctly cited always - see page 10 especially - there is no Figure 3D.

    *It is unfortunately not so easy to correct the language without specific examples. We have however gone through the text carefully, and also asked a native English speaker to assess the language, and corrected accordingly. We are sorry for the Figure mistake, this has now been corrected together with a general update of figure numbers based on some modifications of the manuscript structure. *

    Reviewer #3 (Significance (Required)):

    The advance is not groundbreaking but still interesting and worthy of publishing, if proper controls and better referencing can be done.

    We hope that we after having related all ChIP qPCR data to averaged Cut efficiencies for each strain, and edited the discussion to relate it more appropriately to both new and older correct references, have been able to handle the issues raised and motivate publication of the study.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper presents data analysing the recruitment of Scc2 to double strand breaks. It makes the interesting observation that its recruitment is Tel1 but not Mec1 dependent, and does not require remodelers (it seems). It does correlate with resection but the mechanism of loading is unclear. I have a few issues on controls and alignment of text with results in this manuscript. Also there is some omission of important recent work and some old studies. But if these points can be resolved it could be published.

    Major points:

    1. The cut efficiency under all conditions tested needs to be presented and the CHIP needs to be normalized in every assay to the cut efficiency. This is particularly relevant in the mutants of remodelers as they definitely influence the efficiency of Gal-HO induction. This must be included for every chip result.
    2. The arp8 delta mutant is clearly polyploid and probably has some suppressor mutation or another problem. They should discard the arp8 results and get a proper and controlled arp8 delta strain (from another lab in europe - there are several with good W303 strains).
    3. The text does not accurately reflect the results in several places. For instance .. on page 10 where the result of sgs1 exo1 mutant strain is described, it is said that "Recruitment of Scc2 to the DSB was drastically reduced.... and "consistent with long range resection the effect was less promiment closer to the break.". First, the word "drastic" is not appropriate for a drop of about 50% (on average) and in reality the drop is more significant near the cut (+1kb) than far from the break (+ 10 or 30 kb).... - the data are the opposite of what is stated. and it is not drastic. I do not contest that it correlates with resection, if the HO-cut efficiency is equal in all strains.
    4. The results with INO80 and SWR1 are not really compelling - what is the cut efficiency in these strains. Moreover, the "confusion" in the literature is only because people look at different loci and different conditions. INO80 does affect resection (see Van Attikum et al., 2007; and Cheblal A et al., MOlecular Cell 2020) for resection assays in wt and mutant strains. And it is very strange that the VAn attikum et al., Cell 2004 (the back to back paper with Morrison et al Cell 2004) is not cited. The data on resection is clear in this early work. But it appears that the arp8 mutant used has other mutations and polyploidization, and should clearly be discarded. Nhp10 impact is a bit controversial but not arp8 with a good strain. The references in general are missing Cheblal A et al., Molecular Cell 2020 for Cohesin recruitment, impact on resection and arp8 impact and ditto. Also missing is Deshpande I et al., molecular Cell 2017 for RPA-Ddc2-Mec1 interactions. These omissions are strange and in fact create confusion in the ms.

    Minor points:

    The english usage needs to be corrected at a few places... and figures are not correctly cited always - see page 10 especially - there is no Figure 3D.

    Significance

    The advance is not groundbreaking but still interesting and worthy of publishing, if proper controls and better referencing can be done.

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    Referee #2

    Evidence, reproducibility and clarity

    Cohesin is a key structural component of chromosomes. Amongst its functions, cohesin plays a critical role in ensuring the accurate repair of double stranded DNA breaks (DSBs). Intuitive as this may seem, a number of fundamental open questions remain. One of these questions is, how does the cohesin loading machinery recognise a DSB? This issue is addressed in the present study. The manuscript begins with a well-written introduction into the fields of DSB repair, as well as cohesin. The research aim is clearly laid out. Experiments follow that sequentially investigate known steps of the DSB repair pathway, asking how these steps intersect with the cohesin loading machinery.

    On the positive side, this is a technically very well conducted study (investigating the cohesin loader has proven tricky in many contexts). The study is systematic and explores the known steps during DSB repair for their impact on cohesin loader recruitment. The authors find a surprising separation of function. The DSB pathway up until H2AX phosphorylation and DNA end resection is required for both cohesin loader recruitment, as well as consequently for cohesin loading. The Mec1 checkpoint kinase, in contrast, is dispensable for cohesin loader recruitment but is required for cohesin loading. This suggests that Mec1 supports cohesin loading at a step beyond that of attracting the cohesin loader. The manuscript thus contains important information that will be of interest to a wide range of researchers in the DNA repair and cohesin fields.

    The limitation of the study lies in the fact that the molecular determinant for cohesin loader recruitment to DSBs remains unknown. H2AX phosphorylation and DNA end resection are shown to be prerequisites, but how do these events form a molecular mark that the cohesin loader recognises? And what is this mark? Equally, how does the Mec1 kinase permit cohesin loading additionally to the cohesin loader?

    Specific comments:

    Figure 1. It would be interesting to overlay the Scc2 prolife around the DSB next with that of Scc1 (obtained previously under similar conditions?), to contrast the loading site with the final cohesin distribution.

    Figure 2. Using the same y-axis scale from 1-4 amongst panels A-D could make evaluation of the data easier.

    Figure 3. Panels A and B contain data that are important to interpret the DNA end resection results shown in Figure S2. Maybe that latter data, which conveys the main conclusion from the figure, could be incorporated within the main figure?

    Figure 5. In this figure, the authors begin to investigate possible contributions of candidate cohesin loader receptors, in the form of chromatin remodelling complexes. The Swr1 and INO80 remodellers have an effect on DNA end resection that parallels the effect on Scc2 recruitment, suggesting that their main contribution might be that of facilitating DNA end resection.

    This relationship remains less well documented in the case of Sth1 depletion. Both when using the sth1-3 allele, or degron depletion, the authors observe a relative reduction of cohesin loader recruitment, compared to what they would otherwise expect. However, in both cases a side-by-side analysis of a similarly-treated wild type strain is missing. Whether or not RSC inactivation impacts cohesin loader recruitment therefore remains uncertain. It is also not documented what the corresponding effect of RSC inactivation on DNA end resection might be. Given that previous results suggested that RSC might contribute to cohesin loading at DSBs, the nature of how RSC does this could maybe be clarified before publication.

    Significance

    see above.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript entitled "Recruitment of Scc2/4 to double strand breaks depends on yH2A and DNA end resection", Scherzer et al. study the role of Scc2 in DSB repair in yeast. Scc2 is part of the cohesin loader and it is required for cohesin loading in response to DSB. The authors study the chromatin association of Scc2 by ChIP-qPCR and use genetics to identify factors that affect its recruitment. They show that Scc2 is enriched up to 10 kb from the break site, similar to cohesin and identify MRE, TEL1 and yH2A as important factors for Scc2 chromatin binding. Remarkably, MEC1 that has been shown to regulate cohesin under these conditions is dispensable for Scc2 recruitment. While DNA resection is important for Scc2 recruitment, chromatin remodelers don't play a significant role in it despite numerous reports on their effect on cohesin loading during the cell cycle. The manuscript provides new and important information on cohesin regulation in response to DNA damage.

    Major comments:

    The experiments are done appropriately and contain the required control. The results are presented clearly and with adequate statistics and support the conclusions. The experiments provide valuable information. However, the low resolution of the experimental setup is limiting, and dynamic information of Scc2 binding is lacking. I would agree with the authors that this kind of information may be beyond their scope. However, the absence of this information reduces the overall impact of the manuscript.

    1. ChIP-seq, of at least some of the key experiments, could provide information on the specific Scc2 binding sites and elucidate whether cohesin is translocated from the loading sites or accumulate in its proximity.
    2. It has been suggested that Scc2 and Pds5 are mutually exclusive in cohesin complexes. It would be interesting to check in the current experimental setup (ChIP-qPCR) if Pds5 is mimicing Scc2 pattern

    Minor comments:

    1. Adding a threshold line to the graphs at fold change= 1 (no enrichment in respect to wild type) will increase their readability.
    2. Fig. 1A- Add times to the schematic. Modify the text to GAL addition/break induction.
    3. Page 9. The authors write: "Cohesin failed to be loaded at the DSB in a mec1Δ background (Fig 3A)". However, the figure shows reduced cohesin binding in mec1delata in respect to the wild type.
    4. Page 10. ".......recruitment to the DSB compared to wild type (Fig 3D).". Should be Fig. 4D.
    5. Figure legend 3. "........Protein samples were taken after 3 hours arrest (G2/M, lane 1),....." The benomyl arrest is referred to as G2 arrest in the text but G2/M arrest in the legend. Consistency is needed.
    6. I suggest presenting the suggested model in a figure

    Significance

    I am an expert in cohesin biology.

    The Scc2-Scc4 complex has been identified as an essential factor for cohesin loading during the cell cycle (Ciosk et al., 2000). This function has been shown to be essential for cohesin role in response to DNA DSB (Unal et al., 2004, Storm et al., 2004). The interplay between Scc2 and the cohesin has been studied mostly in the context of the cell cycle. It has been shown that Scc2 activates the ATPase activity of cohesin and promotes its translocation from the loading site. Scc2 and Pds5 are mutually exclusive and their switch suppresses cohesin ATPase activity (Hu et al., 2011, Petela et al., 2011). However, the Scc2-cohesin interplay has been poorly studied in the context of DNA repair. The current work adds valuable information on the factors that recruits Scc2 to the break site and identifies end resection as the key event in this process. This information is novel and important and its contribution to the fields of cohesin and DNA repair should not be overlooked. However, ChIP-seq information can increase the overall impact.

  6. Version published to 10.1101/2021.07.09.451733v1 on bioRxiv