PGENETICS-D-26-00165

A novel tti1 mutation in the TTT complex specifically eliminates the cellular function of Rad3ATR, but not that of other PIKKs in fission yeast

PLOS Genetics

Dear Dr. Xu,

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Dmitry A. Gordenin, Ph.D.

Academic Editor

PLOS Genetics

Giovanni Bosco

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: General Comment

The current model proposes that the Tel2–Tti1–Tti2 (TTT) complex regulates the maturation of all PIKKs. The authors have identified and characterized an interesting separation-of-function mutation, tti1-N18, and propose that this allele is specifically defective in Rad3 kinase function.

However, given the broad role of TTT in PIKK regulation, additional evidence is required to demonstrate that the phenotypes observed in tti1-N18 mutants are uniquely attributable to compromised Rad3 kinase activity rather than to more general defects in PIKK stability, maturation, or signaling. The tti1-N18 mutation could also influence other components within the Rad3 pathway or affect parallel PIKK-dependent processes. Overall, while the proposed model is intriguing, further experimental validation is necessary to establish that this mutation specifically impairs Rad3 function rather than broadly affecting PIKK regulation.

Specific Points

1. Potential involvement of other PIKKs

Because the Tel2–Tti1–Tti2 (TTT) complex promotes the maturation of all PIKKs, it remains possible that the tti1-N18mutation affects additional PIKKs beyond Rad3. The assay presented in Fig. 6 may not be sufficiently sensitive to detect subtle or partial defects in other PIKK pathways. A more comprehensive analysis of multiple PIKK-dependent outputs using different read-outs—such as kinase activity, substrate phosphorylation—would strengthen the claim that tti1-N18 is specifically defective in Rad3 function.

2. Because TTT appears to be destabilized in the tti1-N18 mutant (Fig. 7), it is unclear why the resulting phenotype would be specific to the Rad3 pathway. If TTT function is globally compromised, one might expect defects across multiple PIKK pathways.

One possible explanation is that different PIKKs may vary in their expression levels and turnover rates. For example, if Rad3 is expressed at higher levels for full function or has a faster turnover rate than other PIKKs, it may be more sensitive to partial TTT dysfunction, thereby producing an apparently Rad3-specific phenotype.

Reviewer #2: See attachment

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PloSG-2026-a.docx

PGENETICS-D-26-00165R1

A tti1 mutation in the Tel2-Tti1-Tti2 complex specifically eliminates the cellular function of Rad3ATR, but not that of other PIKKs in fission yeast

PLOS Genetics

Dear Dr. Xu,

Thank you for submitting your manuscript to PLOS Genetics. After careful consideration, we feel that it has merit but does not fully meet PLOS Genetics's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please take care to read carefully review #1 comments on possible interpretation and suggestions regarding Figure 6. I expect that changes in language to address these concerns will be sufficient, and no new data/experiments are required.

Please submit your revised manuscript within by Jun 03 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosgenetics@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgenetics/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Giovanni Bosco, Ph.D.

Section Editor

PLOS Genetics

Giovanni Bosco

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I agree that the tti1-N18 mutation exhibits a much stronger defect in the Rad3 pathway compared to other PIKK pathways, and this phenotype is interesting. However, the revision does not provide a satisfactory explanation for this difference.

The authors argue that, because Tra1 and Tra2 are not kinases, these pathways cannot be analyzed in the same way; however, this point is not fully convincing. Tra1 and Tra2 have well-established roles in transcription, and transcriptional outputs can therefore serve as functional readouts for their activity.

Similarly, Tor1 and Tor2 signaling regulates transcriptional programs, including nutrient-responsive gene expression and ribosome biogenesis. Thus, comparable transcription-based analyses could, in principle, be used to assess defects in these pathways as well. This issue is not addressed in the revision.

In addition, a more careful analysis using higher concentrations of stress-inducing agents would strengthen the conclusions. Notably, even in the cell proliferation assays, the tti1-18 mutant forms smaller colonies in the presence of caffeine, suggesting a measurable growth defect that warrants further investigation (Fig. 6).

Reviewer #2: All my concerns have been addressed in the revised version.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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To enhance the reproducibility of your results, we recommend that authors deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr Xu,

We are pleased to inform you that your manuscript entitled "A tti1 mutation in the Tel2-Tti1-Tti2 complex specifically eliminates the cellular function of Rad3ATR, but not that of other PIKKs in fission yeast" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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PLOS Genetics

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PLOS Genetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I do not have any further questions. However, I note that the serial dilution assays provided may have limited sensitivity for assessing PIKK function, as caffeine and rapamycin primarily slow down cell growth rather than induce cell death. While the authors' interpretation is certainly plausible, additional experiments or supporting evidence could further strengthen the conclusion.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

**********

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Reviewer #1: No

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