PONE-D-24-49625Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function.PLOS ONE

Dear Dr. Weicksel,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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 submit your revised manuscript by Jan 08 2025 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 plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Hari S. Misra, Ph.D.

Academic Editor

PLOS ONE

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“SEW

- P20GM103430

- Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence.

- https://web.uri.edu/riinbre/

- RI-INBRE was not part of the study

JBTW

- R15GM144903-01

- National Institute of General Medical Sciences of the National Institutes of Health

- https://www.nigms.nih.gov/

- NIGMS was not part of the study”

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Additional Editor Comments:

Manuscript is reviewed by 2 subject experts and their comments are appended below for your attention. I have gone through it and concur their comments. Additionally, authors are advised to check the processivity of both the polymerases. Results may help to distinguish if length of polymerization shown in Fig 4, is because of higher processivity of the enzyme or the ability of enzyme to reinitiate in multiple events.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study by Thomas et al, demonstrated that the polymerase domain of polymerase theta (POLQ) from zebrafish and human has a similar function in terms of amplifying damaged DNA strands. The authors employed several conventional biochemical and biophysical approaches to examine the several functional aspects of human POLQ in zebrafish POLQ. Finally, they conclude stating that both human and zebrafish polymerase domains are functional orthologs. While this is a fundamental investigation and enriches our understanding about the POLQ functionality in a popular biological model organism zebrafish, there are several demerits in the technical design of the study, interpretation of results and providing strong supportive evidence to establish their statement. Hence, the manuscript in its present form does not meet the scientific rigor and publication standard of the concerned journal. The authors may consider the following points to further strengthen the manuscript.

Major concerns:

1) It is not clearly mentioned whether full-length zPOLQ was tested and compared to that hPOLQ.

2) In terms of fold-change, how efficient is the zPOLQ compared to hPOLQ?

3) Regarding the lesion bypass capacity of POLQ, the authors only showed thymidine dimer adduct. Can zPOLQ bypass any bulky adduct, oxidative base damage, or nicked strand lesion in the dsDNA region to extend the single-stranded region?

4) What was the basis of choosing different duplex and SS oligo sequences? Are they preferred binding sequences or any random ones?

5) Regarding the participation of zPOLQ in MMEJ, what was the microhomology sequence length using in the in vitro analysis?

6) An in vitro MMEJ reporter-based assay would be ideal to show zPOLQ's possible functional role in MMEJ pathway.

7) None of the experiments had statistical analysis and no mentioning of the number of biological replicates.

8) Without any in-cell assays, it is difficult to ascertain the functional similarities of these two POLQ domains.

Minor concern:

1) Lacking citations of many of the pioneering reports in the field.

2) The manuscript requires improvement in English writing style.

3) Most images are of poor quality.

4) Figure 1 requires domain marking for easy readout by readers.

5) Figure 2A should indicate the band size and exact domain name of the purified protein. Mention purity level of each of human and zebrafish POLQ domains. Total yield is ambiguous as there are other non-specific bands visible in the gel image.

6) Figure 3A, mention the DNA type used in this assay, preferably in the Y-axis.

7)Figure 6 requires statistical qunatification of the activity.

Reviewer #2: The topic of the manuscript entitled “Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function” is a detailed and extensive characterization of the Danio rerio (zebrafish) DNA polymerase theta (θ). It is primary research that fills a large gap in our scientific knowledge about an important enzyme of a major model organism. First, the authors show sequence and structural similarity bioinformatically between the human and zebrafish Pol θ (hPol θ and zPol θ, respectively). Then, they overexpress and purify the polymerase domain of both proteins for biochemical characterization. They use a large number of in vitro methods to study and to compare the DNA binding, primer extension, microhomology-mediated end joining and translesion synthesis activities of the two orthologous proteins. They also examine the effect of various divalent cations on the polymerization activity of these Pol θs. The study is well-designed and thorough. Experiments are performed to a high technical standard and are described in sufficient detail. Conclusions are supported by the data. Nevertheless, there are shortcomings in the data presentation that need improvement.

Major Points:

1. Abstract - The authors emphasize the role of loop regions in key functions of Pol θ. However, they do not have any experimental evidence for the role the loop regions of zPol θ play (They have not performed any assays using Pol θ mutants with deleted or point-mutated loops). Therefore, the abstract should be reworded to reflect the true content of the study, and summarize the findings .

2. Fig 1. Structural modeling of zPol θ. - Is this the full length zPol θ or just the polymerase and exonuclease domains? Amino acid numbers should be included in the legend. In Supplemental figure 1 amino acids 1-799 and 1-744 of hPolQ and zPolQ are aligned. However, in Materials and Methods, line 389 says that “polymerase domain of zebrafish Pol θ (residues 1801-2579)” and also elsewhere in the text they refer to “c-terminal zPol θ” (lines 122 and 200). Perhaps a scheme of the domain structure of zebrafish Pol θ showing amino acid numbers should be included in Figure 1. Similarly, in Table 1 exact residues ‘from - to’ should be given.

3. Fig 3. zPol θ binds tightly to ds DNA – Line 151 says that “products were separated on a 6% non-denaturing gel”, however, line 146 contradicts saying “products were separated on a denaturing gel” (probably incorrect, since line 490 states “Native PAGE”). Furthermore, Fig. 3 shows only the quantitation, not the gel. Since the authors declare in Data Availability that “All relevant data are within the manuscript and its Supporting Information files”, gels should be shown too, either in the main Figure 3, or in the Supplementary file. Moreover, since the experiment should be performed three times, error bars should be included in the graph.

4. Fig 4. zPol θ experiences greater nucleotide extension activity compared to hPol θ. - Line 182 states that “Under steady-state conditions 50 nM hPol θ or zPol θ were preincubated with 200 nM 25/40 dsDNA, however line 159 says (probably incorrectly) that “Under standard steady-state conditions, 200 nM of zPol θ or hPol θ was preincubated with 50 nM 25/40 dsDNA”.

5. Fig 5. zPol θ experiences biphasic burst activity. - Again, like for Fig. 3, it would be nice to see the gels that were quantitated here.

6. Fig. 6 zPol θ is able to perform MMEJ activity - This figure is not perspicuous. Firstly, probably a C is missing from the lower strand, and this way it looks as if there is a G:G mispairing with the upper strand. Secondly, it is not clear how many nucleotides Pol θ adds to the ssDNA. Explanations showing ’n’ and ’n+x’ are missing (In the raw gels file there is a caption “double-stranded DNA”). Interestingly, it looks as if a lot of nucleotides are added, at least 10, even though the templating part in the structure is only 6 nt long. Or is the run different just because this is a native gel, not a denaturing one? The authors could pre-anneal the ssDNA for control. Alternatively, the extended product can be run on a denaturing gel to see the exact size or shorter reaction times could be used to see shorter extended products. Line 229-230 in the main text says “We hypothesize the smaller product bands are indicative of classic snap-back synthesis”. This is not clear either, the authors could mark in the figure which “smaller product bands” they mean.

7. Fig. 7 - Unfortunately, Fig. 7 is missing from the manuscript uploaded to PLOS One site. There is another copy of Fig. 4 here. The intended Fig 7 can be judged from the Supplementary raw gels file. The left part of the gel image (with Mg2+) shows some running anomalies and smears. It is not clear if dCTP is incorporated at all, and whether one or two dGTPs are incorporated opposite the T-T dimer. Moreover, it cannot be established after which nucleotide Pol θs stall in case dATP is added. Since probably all experiments have been repeated 3 times, the authors should provide another gel picture with clearer bands.

8. Fig. 8. - Unfortunately, Fig. 8 is also missing from the manuscript! The intended Fig. 8 can be assessed from the Supplementary raw gels file. There are smears/ running anomalies here again. Line 276 says “hPol θ could incorporate every nucleotide to some extent”, however, it seems as if there was no incorporation of dCTP and dTTP. The authors may want to provide another gel picture with clearer bands.

9. Materials and Methods

Template oligonucleotides are given in 3’ to 5’ direction in lines 464, 467 and 470. This contradicts conventions and the format to show dsDNA will not work in text. Annealed ds substrates could be shown in a table, or may not be necessary, if they are shown in the figures (like Fig. 4).

10. Line 494, mx, x, Y, and b should be specified, explained. Similarly, kobs and kss should be specified in line 508.

11. The Discussion needs improvement in writing. E. g. line 314 can be deleted. The authors could discuss whether polymerases of D. rerio have been biochemically or genetically characterized yet.

Minor Points:

1. The manuscript should undergo extensive language and grammar corrections. For example, “zPol θ experiences greater nucleotide extension activity” should read ‘zPol θ exerts/ exercises/ has/ possesses higher nucleotide extension activity’. “zPol θ catalytic activity similar to other DNA polymerases” should read ‘zPol θ catalytic activity is similar to other DNA polymerases’. e.t.c.

2. The References/ Work Cited does not follow a uniform format. The publication date is given as year (e.g. line 610) or year, month (e.g. line 603), or year, month, day (e.g. line 606) or the publication date is missing (line 613).

3. Supplement 2. “hWT and zWT Pol θ” name is used. Are these proteins the same as the ones in the other figures? If so, they should be called ‘hPol θ and zPol θ’, just like in the other figures.

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Reviewer #1: Yes:  Joy Mitra

Reviewer #2: No

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PONE-D-24-49625R1Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function.PLOS ONE

Dear Dr. Weicksel,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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. Tiff file of Figure 3 seems missing. When we click button for Figure 3, it downloads figure 4 and so on... Please correct it, recheck it carefully before resubmitting.

 Please submit your revised manuscript by Mar 27 2025 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 plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• 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, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Hari S. Misra, Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function.

PONE-D-24-49625R2

Dear Dr. Weicksel,

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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Kind regards,

Hari S. Misra, Ph.D.

Academic Editor

PLOS ONE

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