-->PONE-D-26-13812-->-->A role for nucleosome remodellers during resection of deprotected telomeres in yeast-->-->PLOS One

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Additional Editor Comments:

In addition to the one external reviewer, the AE also reviewed the paper independently. Those comments are incorporated into the comments below.

Major Issues:

1.    With regards to the effects of nucleosome eviction on resection, the authors state that eviction also prevented telomere resection.  However, the authors cannot distinguish between resection of the non-nucleosomal telomeric sequences followed by a block in nucleolytic  progression and a block in resection at the telomere itself since QAOS does not measure telomeres sequences directly. To resolve this issue, the authors should use native gel electrophoresis of telomeric sequences to look for the influence of mutations in RSC and SWI/SNF complexes on the accumulation of telomeric single-stranded character.

2. The epistasis data for the influence of sgs1 and eco1 appears to be misinterpreted. Since the sgs1 mutation alone has no influence, its lack of effect in the double mutant provides no meaningful information. This interpretation should be eliminated from the manuscript.

3. Reviewer 1 makes a valid point that the decrease in the Western blot  of ASISi expression may be over interpreted. For a better interpretation, a more quantitative assessment is require.

Presentation Issue

4. I agree with the reviewer that the QAOS data would be more easily interpretable if the targets were separated and the wild types and mutants were included in the same graph. I suggest that the authors try this configuration to see if this increases clarity.

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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-->5. Review Comments to the Author

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Reviewer #1: In this study, Deiser et al. investigated how exonuclease pathways and chromatin context influence DNA double strand break (DSB) repair by homologous recombination following long-range resection. They employ the cdc13-1 temperature-sensitive allele in S. cerevisiae, which induces deprotected telomeres as previously described. Cdc13 is a component of the CST complex, which binds the single-stranded 3' telomeric overhang and prevents its recognition as a DSB. A critical step in homologous recombination (HR) is DNA end resection, which generates a 3' single-stranded overhang that enables strand invasion. To quantify resection, the authors used QAOS (Quantitative Amplification Of Single-stranded DNA), which relies on primers that preferentially amplify single-stranded 3' telomeric DNA. They show that resection requires the exonuclease Exo1, whereas loss of the helicase Sgs1, which part of an alternative resection pathway, does not measurable affect resection, consistent with previous results by Ngo and Lydall (2010). Using an AID-based degron system, they further demonstrate that Dna2, an essential nuclease that typically acts with Sgs1, is also critical for resection. Combined impairment of Exo1 and Dna2 resulted in an almost complete loss of resection. The authors also examine the contribution of the histone variant H2A.Z and the nucleosome remodelers RSC and SWI/SNF to telomere resection. Combined loss of H2A.Z (encoded by HTZ1) and the catalytic subunit of the SwrC complex did not produce detectable changes in resection. This result is different from observations at other genomic sites, where DSBs are induced by an endonuclease and resection is significantly impaired in the htz1 swr1 mutant. In contrast, loss of either Sth1 (RSC) or Snf2 (SWI/SNF) nearly abolished resection in the cdc13-1 system, underscoring the importance of nucleosome remodeling for resection, consistent with other genomic locations.

Overall, the experiments are carefully performed, include sufficient biological controls, and the data are generally convincing and critically discussed. For the most part, the conclusions are supported by the presented results (see major points). Although the cdc13-1 system has previously been used to study DSB repair at deprotected telomeres, this study extends prior work by investigating the roles of Dna2 and the nucleosome remodelers RSC and SWI/SNF, which are difficult to examine due to their essential functions. The authors further propose that the cdc13-1 system can serve as a general model for studying resection, as it appears to recapitulate key features of DNA repair. However, the data also reveal context-dependent differences; for instance, H2A.Z contributes to resection at endonuclease-induced DSBs but does not appear to play a comparable role in resection near telomeres. Moreover, although Dna2 typically functions in conjunction with Sgs1, loss of SGS1 does not measurably affect long-range resection in this system. Therefore, some of the broader conclusions, particularly regarding the general applicability of the cdc13-1 system, should be tempered to reflect these context-dependent differences. Despite these limitations, the study provides a useful framework for dissecting chromatin-dependent regulation of resection in a defined telomeric context.

Major

1. It is very difficult to compare QAOS resection data between WT and mutants across separate panels. Plotting WT and mutants within the same graph, while keeping target loci separate, would facilitate direct comparison and improve statistical evaluation.

2. I am not convinced that the data support the conclusion that Dna2 acts in conjunction with Sgs1/STR during telomere resection. The authors show that loss of Sgs1 does not affect resection, either alone or in combination with loss of Exo1. This absence of a detectable phenotype contrasts with the reduced resection observed in exo1 mutant and in the dna2 degron allele. Taken together, these results suggest that Sgs1 does not make a substantial contribution to resection in this context, consistent with the previous report by Ngo and Lydall 2010. Accordingly, the conclusion that the sgs1 mutant is epistatic to the dna2 degron strain is not supported by the current data. In the strict sense, epistasis analysis requires that both single mutants display a phenotype, allowing non-additive effects to be assessed. The corresponding statements in the Abstract and Results should therefore be revised to reflect these limitations.

3. While the authors observe reduced resection in the swr1∆ htz1∆ double mutants, they argue that this may be indirect, as ASiSI expression levels are also decreased in this strain background. Although I appreciate this critical assessment, I'm not fully convinced that the moderate change in AsiSI expression based on the western blot data shown in Figure S3B fully explains the nearly complete absence of resection in swr1∆ htz1∆. Although AsiSI -3HA expression levels vary, the non-specific band used as a loading control also show variability (although signal intensities appear weaker), which complicates interpretation. Without quantitative analysis of western blot signals, including serial dilutions to properly assess quantitative differences in signal intensities, differences in protein levels cannot be reliable assessed. Alternatively, AsiSI expression could be quantified by RT-qPCR, which may provide greater sensitivity. Addressing this limitation would help distinguish whether H2A.Z plays distinct roles at deprotected telomeres and other genomic locations induced by endonucleases.

Minor

1. The time points in the prolonged time course experiments (e.g. 2 and 4h) seem to display less ssDNA accumulation than the corresponding 2 h time points in Figure 2E (Y5000). The authors should comment on this discrepancy.

2. Did the authors assess whether Fun30 contributes to resection at de-protected telomeres? If not, please clarify the rationale for not testing it.

3. The authors should explain the rationale why they used the htz1 swr1 double mutant instead of single mutants.

4. Mentioning of CST and some additional information (histone variant -> H2A.Z, exonuclease -> Exo1, helicase -> Sgs1) in the abstract could improve accessibility for readers less familiar with the topic.

5. Author list: There appears to be a spelling error in the first author's name

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Reviewer #1: No

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A role for nucleosome remodellers during resection of deprotected telomeres in yeast

PONE-D-26-13812R1

Dear Dr. Pfander,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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-->6. Review Comments to the Author

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Reviewer #1: For the most part, all of my previous concerns have been addressed. The authors have effectively resolved the points that were previously unclear through improved data presentation, additional experiments, and revisions throughout the manuscript.

While I still believe that presenting the QAOS plots for individual targets and displaying the wild type, single mutants, and double mutants side by side within the same figure would provide a more effective comparison, the revised arrangement adopted by the authors substantially improves the presentation and facilitates comparison of the data.

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Reviewer #1: No

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