Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article addresses an interesting and relevant topic: using the model organism C. elegans to study how mutations in different regions of the smc-6 gene affect SMC-6 function. The authors used EMS-induced mutagenesis in a C. elegans strain carrying an nse-1::gfp reporter to find mutants with altered NSE-1::GFP localization. Through genetic crosses and PCR–RFLP analysis, they mapped the causal locus to chromosome II in a region containing the smc-6 gene. Whole-genome sequencing then revealed smc-6 mutations in three mutants: two nonsense alleles and one missense variant in a conserved region of SMC-6. The authors next performed functional assays using several DNA-damaging agents at different stages of C. elegans development to characterize the effects of these mutations.

However, the interpretation of the mutations is not sufficiently supported by the data. The main conceptual problem is that the three alleles are presented as an allelic series suitable for structure–function analysis and as evidence for a domain hierarchy model for ATPase head versus hinge. In reality, two of these alleles introduce premature stop codons in exon 2 and exon 3 of smc-6, a gene with seven exons, which would be expected to trigger nonsense-mediated mRNA decay and thus behave as strong loss-of-function, essentially null, mutations. The authors treat these nonsense alleles as if they specifically modify ATPase head function, like a rationally designed missense mutation within the head domain. This is not a realistic interpretation for such early truncations, which are unlikely to preserve any intact ATPase head. In this context, it is also misleading to show sequence alignments to assess the conservation of an amino acid that is mutated to a stop codon, or to illustrate how this residue interacts with other components of the SMC5/6 complex.

To strengthen their conclusions, I recommend that the authors directly assess smc-6 expression in the nonsense mutants, for example by measuring smc-6 mRNA levels (RT–qPCR or RNA-seq) and, if possible, SMC-6 protein levels. Such data would clarify whether the nonsense alleles mainly act through nonsense-mediated mRNA decay and strongly reduced SMC-6 dosage, or whether truncated proteins are produced. This would allow a more accurate classification of these alleles and help to adjust the claims about an allelic series and a domain-specific head-versus-hinge hierarchy accordingly.

Minor issues:

Figure 2: Not all PCR–RFLP markers on LGII designated by red numbers showed differences between mutant and control pools.

Figure 3: It is unclear what the lowercase letters a, b, and c above the lanes indicating recombination events signify. It appears that these labels denote sample numbers rather than marker order (cM).

Section 2.6: The text describes the results for only two of the mutants, whereas Figure 5 shows results for all three mutants generated by the authors.

Section 4.6: If the authors used a standard published 48‑marker panel unchanged, they should cite the source or add a small table (main or supplementary) listing, for each marker, chromosome, genomic coordinate (WS build), primer sequences, and expected fragment sizes after DraI digestion; the same applies to the SNP markers used for chromosome II. The commercial provider for WGS should also be indicated, along with the sequencing platform and the reference genome build.

Author Response

Dear Reviewer,

Thank you very much for your time, careful evaluation, and constructive comments on our manuscript entitled “Allelic analysis of smc-6 reveals domain-specific roles in DNA repair in Caenorhabditis elegans” (Manuscript ID: ijms-4218308). We have carefully considered each comment and revised the manuscript accordingly. In the revised version, all changes in the manuscript are highlighted in red, and our point-by-point responses to the reviewers’ comments are provided below in blue for convenience. Detailed corrections and explanations are listed point by point below.

 

We would be grateful if you could reconsider our manuscript for review and provide constructive feedback.

 

If you have any questions, please do not hesitate to let us know. Thanks, and Best regards.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript represents a continuation of a series of studies investigating the SMC-5/6 complex and its role in genome stability and DNA repair within the Caenorhabditis elegans model. Following major revisions, the manuscript will be suitable for publication in a special issue of the International Journal of Molecular Sciences “DNA Damage and Repair: Current Research”.

1. It remains unclear why the manuscript does not cite several works that are directly relevant to the current study:

• Wolters S., Ermolaeva M.A., Bickel J.S., Fingerhut J.M., Khanikar J., Chan R.C., Schumacher B. Loss of Caenorhabditis elegans BRCA1 promotes genome stability during replication in smc-5 mutants. Genetics. 2014; 196(4):985-99. doi: 10.1534/genetics.113.158295;
• Odiba A.S., Ezechukwu C.S., Liao G., Li S., Chen Z., Liu X., Fang W., Jin C., Wang B. Loss of NSE-4 Perturbs Genome Stability and DNA Repair in Caenorhabditis elegans. J. Mol. Sci. 2022; 23(13):7202. doi: 10.3390/ijms23137202;
• Odiba A.S., Liao G., Ezechukwu C.S., Zhang L., Hong Y., Fang W., Jin C., Gartner A., Wang B. Caenorhabditis elegans NSE3 homolog (MAGE-1) is involved in genome stability and acts in inter-sister recombination during meiosis. Genetics. 2023; 225(2):iyad149. doi: 10.1093/genetics/iyad149;
• Odiba A.S., Ezechukwu C.S., Liao G., Hong Y., Fang W., Jin C., Gartner A., Wang B. SMC-5/6 complex subunit NSE-1 plays a crucial role in meiosis and DNA repair in Caenorhabditis elegans. DNA Repair (Amst). 2024; 137:103669. doi: 10.1016/j.dnarep.2024.103669;
• Yuan H., Odiba A.S., Liao G., Zhou Z., Fang W., Jin C., Li S., Liu X., Wang B. Separation-of-Function Alleles of smc-5 Reveal Domain-Specific Defects and a Conserved Residue Critical for Genome Maintenance. Biomolecules. 2025; 15(6):755. doi: 10.3390/biom15060755;
• Toraason E., Salagean A., Almanzar D.E., Brown J.E., Richter C.M., Kurhanewicz N.A., Rog O., Libuda D.E. BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans Elife. 2024; 13:e80687. doi: 10.7554/eLife.80687.

 

2. Both the Introduction and Discussion sections require significant revision. Important references are missing from the context of the work presented.
3. Reference 11 refers to a study focusing primarily on the NSE1-3-4 subcomplex in budding yeast, rather than specifically on the NSE1 subunit. Please include citations that specifically discuss the Nse1 subunit. Such publications are available and would provide a clearer rationale for selecting this particular subunit over NSE3 or NSE4. At present, the introduction does not provide any rationale as to why NSE1 was chosen.
4. Both the Introduction and Discussion sections present pooled data on the SMC-5/6 complex from various model organisms without sufficient distinction. Please provide species-specific details and clearly indicate what is currently known about the SMC-5/6 complex specifically in Caenorhabditis elegans.
5. Please provide definitions for all abbreviations used in the manuscript and include them in a separate “Abbreviations” section.
6. Please use the full terminology instead in the Abstract (e.g., provide the full name for the SMC-5/6 complex and EMS at their first mention).
7. Please ensure consistent formatting throughout the manuscript. For example, the complex is abbreviated as “SMC-5/6” in the Abstract and Keywords, but appears as “Smc-5/6” in the Introduction.
8. The quality of Figure 3 is poor, making it unreadable. Both Figure 3 and Figures 4c–e require higher resolution.
9. In Figure 5, some comparison lines in panels (c)–(e) are indistinct.
10. Figure 6. There is a typo in the labels to graphs (g)–(k): “cisplatin” should be used instead of “cirplatin”. There is a missing space in the figure caption after the sentence “Genotoxic stress induces dose-dependent developmental delay in smc-6 mutants.”

 

The Materials and Methods section needs revision.

11. Please add a reference to a specific methodology for preparing NGM plates and M9 buffer for Caenorhabditis elegans (section 4.1).
12. Please provide standard conditions for DNA repair pathway control mutants (section 4.1).
13. Please provide a link to a publication or materials confirming the validation of the obtained transgenic reporter strain nse-1::gfp (section 4.2). How exactly was this strain obtained? Please also provide a reference to the production method.

Section 4.5 describes whole-genome sequencing that insufficient for replicated (only a commercial vendor is listed). Please provide details (libraries, instrument model, read parameters, coverage depth, software, etc.).

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you very much for your time, careful evaluation, and constructive comments on our manuscript entitled “Allelic analysis of smc-6 reveals domain-specific roles in DNA repair in Caenorhabditis elegans” (Manuscript ID: ijms-4218308). We have carefully considered each comment and revised the manuscript accordingly. In the revised version, all changes in the manuscript are highlighted in red, and our point-by-point responses to the reviewers’ comments are provided below in blue for convenience. Detailed corrections and explanations are listed point by point below.

 

We would be grateful if you could reconsider our manuscript for review and provide constructive feedback.

 

If you have any questions, please do not hesitate to let us know. Thanks, and Best regards.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have read all the comments and made the necessary edits. However, please note a few additional details. 1. Please highlight "in vivo" in italics (line 14, 47, 56, 58, 70, 80, 88, 382, 399, 460, 481). 2. The resolution of Figure 4 should be increased as the labels are not legible.

Author Response

Dear Reviewer,

We sincerely thank you for your careful re-evaluation of our revised manuscript and for your positive comments. We are truly grateful that you found the comments from the previous round had been addressed and that the necessary revisions had been made. We also greatly appreciate your additional suggestions, which have helped us further improve the manuscript's clarity and overall presentation.

 

In response to your comments, we have carefully revised the manuscript accordingly. The specific revisions and our point-by-point responses are provided below. For the reviewer’s convenience, all revisions made in the manuscript are highlighted in red, and our responses are presented in blue.Please find our detailed responses below.

 

We would be grateful if you could reconsider our manuscript for review and provide constructive feedback.If you have any questions, please do not hesitate to let us know. Thanks, and Best regards.

Author Response File: Author Response.pdf