Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

Fft3, a member of the SNF2 family of ATPase-dependent enzymes, plays a dual role at DNA replication barriers in fission yeast by promoting both DNA resection and restart of blocked DNA replication forks.

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Sincerely, Andrea Leibfried, PhD Executive Editor Life Science Alliance Meyerhofstr. 1 69117 Heidelberg, Germany t +49 6221 8891 502 e a.leibfried@life-science-alliance.org www.life-science-alliance.org A. THESE ITEMS ARE REQUIRED FOR REVISIONS --A letter addressing the reviewers' comments point by point.
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B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** The manuscript by Ait-Saada et al. documents the function of Fft3 in resection at DNA ends in fission yeast. While the function of its homologs in other fungi and in human is known, the role of Fft3 in fission yeast remined unknown. Here they show that Fft3 is important for resection at DNA break ends and at blocked replication forks. The data are convincing and important in the field of DNA repair. I have only few minor comments that need to be addressed: All questions relate to Fig. 1B-D 1. Fft2 has more of his-leu-cells compared to WT. Is this difference significant. Do these also represent SSA event but Leu-or these are NHEJ.
2. His-Leu-colonies in fft3 are SSA products that remain Leu-. Are these stably Leu-? If these are stably Leu-, Sanger sequencing should be done to make sure there is no mutation at LEU2 sequence that occurred during SSA.
3. To make sure that SSA is slower in fft3 it would be good to show representative Southern blot to demonstrate that DSB induction is similar in fft3 and wild type cells. Slower SSA may result from slow resection or slow DSB induction in fft3.
Reviewer #2 (Comments to the Authors (Required)): This is a nice little paper showing the role of the Fft3 SMARCAD homologue in fission yeast in replication fork stability and restart. The authors show that the related Fft1 and Fft2 proteins do not share this role. The experiments are well done and the data are generally convincing. The one concern I have is that the only evidence for delayed resection is using 2D gels. I would like to see an alternative assay for resection with more molecular detail to confirm this observation. Otherwise, I have no significant concerns .
Minor points: it would be helpful to have an English speaker go over the MS as there are minor grammatical issues.
Reviewer #3 (Comments to the Authors (Required)): Anissia Ait-Saada, Olga Khorosjutina, Jiang Chen, J. Peter Svensson, Sarah Lambert, Karl Ekwall Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks Fission yeast Fun30/Smarcad1 family of SNF2 ATPase is involved in histone turnover during transcription and DNA replication in vivo. Ait-Saada et al showed that only Fft3 could have a function in DNA repair utilizing growth assay on MMS plate, DSB repair assay, and RI analysis. Importantly, ATPase activity is required to promote cell resistance to replication stress, indicating that chromatin remodeling activity of Fft3 controls DNA repair process by SSA. Finally, Ait-Saada et al demonstrated a dual role for Fft3 at stalled replication forks. That is, ATPase of Fft3 is necessary for resection and is not necessary for HR-mediated fork restart. Although we already know about Fun30's involvement in resection but we do not know the details about DNA repair process. Interestingly, Ait-Saada et al dissected the important point of Fun30 at blocked DNA replication forks. Overall, authors showed very interesting data and The results presented in the paper are rigorous. Before publication, I may add some suggestions below about their manuscript. 1. Figure 3: the corresponding dot plots have to be overlaid in the bar charts since the apparent error bars are too high.
2. Figure 4: It is interesting that K318R still showed comparable RS Frequency. Is there any possibility that other remodeler can be involved in this particular step? Or other Fun30 ortholog?

Point by point response to reviewer's comments
We thank all three reviewers for constructive criticisms that have helped to improve this manuscript. Below is our response to all the specific points.
Reviewer #1 (Comments to the Authors (Required)): The manuscript by Ait-Saada et al. documents the function of Fft3 in resection at DNA ends in fission yeast. While the function of its homologs in other fungi and in human is known, the role of Fft3 in fission yeast remined unknown. Here they show that Fft3 is important for resection at DNA break ends and at blocked replication forks. The data are convincing and important in the field of DNA repair. I have only few minor comments that need to be addressed: All questions relate to Fig. 1B-D 1. Fft2 has more of his-leu-cells compared to WT. Is this difference significant. Do these also represent SSA event but Leu-or these are NHEJ. Answer: Yes the number of His-Leu-colonies is significantly higher for fft2D compared to wild type (unpaired t-test p=0.036). However these colonies were not sequenced so we can't say if they are SSA products or not.
2. His-Leu-colonies in fft3 are SSA products that remain Leu-. Are these stably Leu-? If these are stably Leu-, Sanger sequencing should be done to make sure there is no mutation at LEU2 sequence that occurred during SSA. Answer: As mentioned on page 4 (second paragraph) sixteen His-Leu-colonies (11 from fft3D and 5 from exo1D) were sequenced and verified to have an intact LEU2+ gene. Therefore they can't be stably Leu-caused by a mutation in the LEU2+ gene.
3. To make sure that SSA is slower in fft3 it would be good to show representative Southern blot to demonstrate that DSB induction is similar in fft3 and wild type cells. Slower SSA may result from slow resection or slow DSB induction in fft3. Answer: We have measured DSB in the mutants during a time course by QPCR. We observe lower DSB induction levels for fft3Δ strains compared to wild type and fft2Δ strains after 5 hours of DSB induction. Therefore, as pointed out by this reviewer, it is possible potentially slower DSB formation in his3-HO region could contribute to SSA products formation. However, due to design and calculations of SSA assay ( Figure 1D), this seems unlikely. The SSA assay shows the kinetics of SSA product formation, relative to an arbitrarily chosen time point (24 hours in SSA assay). This also means only cells with induced DSB are being evaluated. For this reason it does not matter that much if at the start of an assay there are different amount of DSB events as long as sensitivity of detection method used (qPCR) allows to follow product accumulation. Thus, we argue that there is no direct connection between slower SSA and slow DSB induction in fft3Δ and only slow resection is contributing into SSA product kinetics. Slow DSB induction in fft3Δ can result for instance from different expression levels of MATa HOendonuclease which induces DSB in his3-HO region, due to function of fft3 in transcription regulation.
We have included the DSB induction data as Supplementary Figure 2. See also text changes in results and methods sections.

Reviewer #2 (Comments to the Authors (Required)):
This is a nice little paper showing the role of the Fft3 SMARCAD homologue in fission yeast in replication fork stability and restart. The authors show that the related Fft1 and Fft2 proteins do not share this role. The experiments are well done and the data are generally convincing. The one concern I have is that the only evidence for delayed resection is using 2D gels. I would like to see an alternative assay for resection with more molecular detail to confirm this observation. Otherwise, I have no significant concerns . Answer: As an alternative assay, we have analyzed the binding of RPA to the RFB by ChIP-qPCR, that reflects the formation of ssDNA (Tsang et al. J. Cell Science 2014). The recruitment of RPA upstream from the RTS1-RFB was significantly reduced in fft3 cells and in cells expressing Fft3-K148R. This was particularly pronounced from 400 bp and more behind the arrested fork, indicating a less efficient long-range resection. These data have been added as a new panel on figure 2 (Panel E). We believe that this alternative assay provides convincing molecular details to establish that Fft3 and its remodeling chromatin function are necessary to promote the resection of newly replicated strands at arrested forks.
Minor points: it would be helpful to have an English speaker go over the MS as there are minor grammatical issues. Answer: We have corrected grammatical errors.

Reviewer #3 (Comments to the Authors (Required)):
Anissia Ait-Saada, Olga Khorosjutina, Jiang Chen, J. Peter Svensson, Sarah Lambert, Karl Ekwall Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks Fission yeast Fun30/Smarcad1 family of SNF2 ATPase is involved in histone turnover during transcription and DNA replication in vivo. Ait-Saada et al showed that only Fft3 could have a function in DNA repair utilizing growth assay on MMS plate, DSB repair assay, and RI analysis. Importantly, ATPase activity is required to promote cell resistance to replication stress, indicating that chromatin remodeling activity of Fft3 controls DNA repair process by SSA. Finally, Ait-Saada et al demonstrated a dual role for Fft3 at stalled replication forks. That is, ATPase of Fft3 is necessary for resection and is not necessary for HR-mediated fork restart. Although we already know about Fun30's involvement in resection but we do not know the details about DNA repair process. Interestingly, Ait-Saada et al dissected the important point of Fun30 at blocked DNA replication forks. Overall, authors showed very interesting data and The results presented in the paper are rigorous. Before publication, I may add some suggestions below about their manuscript.
1. Figure 3: the corresponding dot plots have to be overlaid in the bar charts since the apparent error bars are too high. Answer: A new version of Figure 3 has been produced with dot plots as requested.
2. Figure 4: It is interesting that K318R still showed comparable RS Frequency. Is there any possibility that other remodeler can be involved in this particular step? Or other Fun30 ortholog? Answer: We have tested the role of other Fun30 orthologues. Combining the deletion of fft1 with fft3 or fft3-K418R mutation did not increase the cell sensitivity to MMS and did not impact the RS frequency. These data exclude a role for Fft1 in promoting fork-restart in the absence of the chromatin remodeling activity of Fft3 (see figure below). Combining the deletion of fft2 with fft3 or fft3-K418R mutation resulted in a severe growth defect that prevents us to apply the RS assay in these strains (see figure below). Also, this synthetic sickness may indicate a role for Fft2 in the absence of Fft3 and its ATPase activity, we cannot further test this hypothesis. Since the data obtained are negative or not fully conclusive, we did not add them to the current manuscript but we can provide them if the reviewer wish them to be included. We did not investigate the role of other chromatin remodeler in the time-frame of this reviewing. Thank you for submitting your revised manuscript entitled "Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks". We would be happy to publish your paper in Life Science Alliance pending final minor revisions, mainly necessary to meet our formatting guidelines: -Please address the remaining comment of reviewer #1 -Please upload the manuscript file in docx format -Please add a callout in the manuscript text to figure 3E -I noticed that the % of DSB repair in figure 1C is based on two biological replicates. It would be good to show the individual bar graphs next to each other instead of the average.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The revised manuscript is suitable for publication however the questions were not completely addressed. This reviewer is convinced that Fft3 promotes resection. This is the main message of the work. Somewhat side observation with SSA products that lead to Lue-colonies could be explained better. The authors state in response to my question #2 (pasted below) that Leucolonies cannot be stably Leu-. How was it tested? Why is is not stated in the main manuscript? Sequencing is not the test for Leu2 expression. Was the phenotype tested? Re-streaked colonies were Leu+? 2. His-Leu-colonies in fft3 are SSA products that remain Leu-. Are these stably Leu-? If these are stably Leu-, Sanger sequencing should be done to make sure there is no mutation at LEU2 sequence that occurred during SSA. Answer: As mentioned on page 4 (second paragraph) sixteen His-Leu-colonies (11 from fft3D and 5 from exo1D) were sequenced and verified to have an intact LEU2+ gene. Therefore they can't be stably Leu-caused by a mutation in the LEU2+ gene.