Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
This study elucidates the molecular linkage between the mobilization of damaged rDNA from the nucleolus to its periphery and the subsequent repair process by HDR. The authors demonstrate that the nucleolar adaptor protein Treacle mediates rDNA mobilization, and the MDC1-RNF8-RNF168 pathway coordinates the recruitment of the BRCA1-PALB2-BRCA2 complex and RAD51 loading. This stepwise regulation appears to prevent aberrant recombination events between rDNA repeats. This work provides compelling evidence for the recruitment of the Treacle-TOPBP1-NBS1 complex to rDNA DSBs and demonstrates the critical role of MDC1 in the rDNA damage response. There are some issues with the over-interpretation of results as described subsequently. Some aspects could be strengthened, for example, a potential role of the RAP80-Abraxas axis, the origin of the repair synthesis (HDR vs. NHEJ), and a direct comparison of the RNF8 and RNF168 recruitment in the absence or presence of MDC1.
We thank the reviewer for the positive assessment of our work and for the constructive suggestions. We agree that certain aspects of the manuscript required clarification, in particular the potential contribution of the RAP80–ABRAXAS pathway, the interpretation of the repair synthesis assay, and the role of RNF168 recruitment. We have addressed these points experimentally where feasible and have revised the manuscript accordingly to avoid overinterpretation.
Reviewer #1 (Recommendations for the authors):
Major comments
(1) In Figures 4C, 4D, and S4B-D, BRCA1 and RAD51, recruitment to nucleolar caps is partially reduced upon RNF168 depletion. Despite this, the authors broadly conclude that recruitment mainly depends on the MDC1-RNF8-RNF168 pathway. Since the RAP80-Abraxas pathway may also contribute, as briefly mentioned in the Discussion, siRNA knockdown of Abraxas would help clarify the relative roles of these two pathways.
We thank the reviewer for this important suggestion. To directly address the potential contribution of the RAP80–ABRAXAS pathway, we depleted RAP80 by siRNA and analysed BRCA1 and RAD51 recruitment to nucleolar caps following I-PpoI-induced rDNA damage.
Strikingly, RAP80 depletion strongly impaired the formation of both BRCA1 and RAD51 nucleolar caps in two independent cell lines (U2OS and RPE1) (new Figure 5). These findings demonstrate that the RAP80–ABRAXAS pathway plays a critical role in BRCA1 recruitment at nucleolar caps.
Together with our observation that RNF168 depletion only partially reduces BRCA1 and RAD51 recruitment, these results indicate that both RNF168-dependent and RAP80–ABRAXAS-dependent pathways contribute to HDR factor recruitment downstream of RNF8-mediated chromatin ubiquitylation.
We have revised the model Figure (Figure 9) and the Results and Discussion sections accordingly to reflect this dual-pathway model and to avoid overemphasising the contribution of RNF168.
(2) In Figure 7C, the EdU-γH2AX PLA assay detects DNA synthesis at rDNA breaks, but it remains unclear whether this signal reflects HDR- or NHEJ-mediated repair. Since MDC1 functions upstream of the DSB repair pathway choice, the observed reduction in PLA signal upon MDC1 depletion does not necessarily reflect impaired HDR alone. Synchronizing cells in G2 or using cell cycle markers would help clarify the repair context and strengthen the interpretation.
We thank the reviewer for raising this important point. We agree that the EdU–gH2AX PLA assay does not exclusively report on HDR-mediated DNA synthesis and may also capture other forms of repair-associated DNA synthesis.
In the revised manuscript, we have therefore tempered our interpretation and now describe this assay more cautiously as a readout of DNA synthesis at sites of rDNA damage, rather than as a direct measure of HDR activity.
Importantly, our conclusion that MDC1 promotes HDR factor recruitment at nucleolar caps is based primarily on the reduced accumulation of BRCA1, PALB2, and RAD51, which are well-established markers of HDR. The PLA assay is now presented as supportive evidence for ongoing DNA synthesis at these sites rather than as a definitive indicator of HDR.
We agree that further experiments, such as cell cycle synchronization or the use of phase-specific markers, would help to more precisely define the repair context, and we have included this point in the Discussion.
(3) The authors propose that MDC1 is essential for RNF8-RNF168 recruitment, specifically at nucleolar rDNA breaks. A side-by-side comparison of RNF8 or RNF168 localization in the presence and absence of MDC1, with IR-treated conditions, would provide important validation of this model. Including representative images in Figure S2C would further support the claim.
We agree with the reviewer that a direct analysis of RNF8 and RNF168 recruitment in the presence and absence of MDC1 would provide valuable mechanistic insight. We therefore attempted to address this experimentally.
However, despite testing multiple antibodies, we were unable to obtain specific and reproducible signals for RNF8 and RNF168 at nucleolar caps, precluding a reliable analysis of its recruitment under these conditions.
Given this technical constraint, we have revised the manuscript to avoid overinterpretation regarding direct RNF168 recruitment and instead focus on functional readouts of downstream ubiquitylation-dependent signalling, such as BRCA1 and RAD51 accumulation.
We note that the requirement for MDC1 in BRCA1 and RAD51 recruitment at nucleolar caps is consistent with a role of MDC1 upstream of RNF8-dependent chromatin ubiquitylation, in line with its established function at IR-induced DSBs.
Minor comments:
(1) The legend for Figure 8 should more clearly explain the proposed mechanism and include concise titles or descriptions for each sub-panel.
We agree with the reviewer that the model should be described in the Figure legend. We have thus updated the model to accommodate the new data and wrote a legend that concisely explains the proposed model. We do not think that titles for each sub-panel are required. Instead, we separately referred to the sub-panels in the legend.
(2) Typos:
(a) Page 8: PRE1 MDC1, as "RPE1 MDC1;
(b) S3 Figure legend: Dhermacon";
(c) Page 29: "80.103"-please clarify or correct.
We thank the reviewer for pointing out these errors. These have been corrected in the revised manuscript
Reviewer #2 (Public review):
Summary:
DNA double-strand breaks (DSB) in repeated DNA pose a challenge for repair by homologous recombination (HR) due to the potential of generating chromosomal aberrations, especially involving repeats on different chromosomes. This conceptual caveat led to a long-held notion that HR is not active in repeated DNA, which was disproven in groundbreaking work by Chiolo showing in Drosophila that DSBs in pericentromeric repeats are mobilized to the nuclear periphery for repair by HR. A similar mechanism operates in mouse cells, as shown by the Gautier laboratory, but the mobilization goes to the nucleolar periphery, called nucleolar caps. In this manuscript, the authors reexamine the role of MDC1 in the mobilization of DSBs in rDNA in human cells. Previous work has shown that MDC1 is replaced by Treacle, the gene associated with Treacher Collins syndrome 1, in its role as the main adaptor of the DNA damage response, and these results are confirmed here. The novelty of this contribution lies in the discovery that MDC1 is required downstream in the recruitment of BRCA1 and RAD51 to nucleolar DSBs that were mobilized to the nucleolar cap. Using multiple MCD knockout models and DSBs induced by the nuclease PpoI, which cleaves at nuclear sites as well as in the 28S rDNA, convincingly documents this role of MDC1 and shows that it acts upstream of the RNF8-RNF168 ubiquitylation axis. Using a proxy assay of co-localization of EdU incorporation at DSBs (gammaH2AX), evidence is provided that MDC1 is required for HR in rDNA. MDC1 was not required for RAD51 recruitment to IR-induced foci, but it is unclear whether this is related to the different DSB chemistry (enzymatic versus IR) or to the localization of the DSB (rDNA versus unique sequence genome).
Strengths:
(1) The manuscript is well-written, and the experimental evidence is nicely presented.
(2) Multiple MDC1 knockout models are used to validate the results.
(3) Convincing back-complementation data clarify the relationship between MDC1 and RNF8.
Weaknesses:
(1) The recruitment of BRCA2 was not directly demonstrated. This caveat could be recognized, as IF for BRCA2 is challenging.
(2) PpoI also induces DSBs in the non-rDNA genome. These DSBs would be an ideal control to establish nucleolar specificity of the events described and clarify whether the difference between IR and PpoI is the chemical structure of the DSB or the location of the DSB.
We thank the reviewer for the positive and insightful evaluation of our work. We appreciate the recognition of the conceptual advance and the robustness of our experimental approaches. We have carefully considered the reviewer’s suggestions and have revised the manuscript to clarify interpretation where appropriate, particularly regarding BRCA2 recruitment and the specificity of I-PpoI-induced DNA damage. Where possible, we have also added new analyses to strengthen the conclusions.
Reviewer #2 (Recommendations for the authors):
(1) The claim that the BRCA1-PALB2-BRCA2 is recruited (abstract, end of results section, discussion page 15) should be qualified as BRCA2 recruitment was not directly demonstrated.
We thank the reviewer for this important point. We agree that BRCA2 recruitment was not directly demonstrated in our study, as reliable immunofluorescence detection of BRCA2 remains technically challenging.
We have therefore revised the manuscript throughout (Abstract, Results, and Discussion) to avoid overstatement and now refer more precisely to the recruitment of BRCA1, PALB2, and RAD51, rather than implying direct recruitment of a BRCA1–PALB2–BRCA2 complex.
We note that BRCA2 function is supported indirectly by the observed RAD51 loading, which depends on BRCA2 activity. However, we have clarified this point to ensure that our conclusions remain fully supported by the presented data.
(2) The temporal sequence established in Figure 1, 1hr BRCA1 and 2 hrs PALB2, argues against recruitment of a stable BRCA1-PALB2-(BRCA2) complex. This should be acknowledged.
We thank the reviewer for this insightful observation. We agree that the temporal separation between BRCA1 accumulation (1 h) and PALB2/RAD51 recruitment (2 h) argues against the recruitment of a pre-assembled, stable BRCA1–PALB2–BRCA2 complex.
We have revised the manuscript to reflect this interpretation and now describe the recruitment of HDR factors as a sequential process rather than as the assembly of a pre-formed complex. This is consistent with current models in which BRCA1 promotes subsequent PALB2 and BRCA2 recruitment, ultimately leading to RAD51 loading.
(3) The model predicts that MDC1-KO cells are proficient for transcriptional repression after nucleolar DSB induction. Has this been tested?
We did not specifically test this in the current work, but previous results published by our group revealed that siRNA-mediated depletion of MDC1 in human cells had a minimal effect on rDNA transcriptional inhibition after DNA damage (Larsen et al., 2024).
(4) The nuclear PpoI DSBs could be analyzed as a specificity control, and clarify whether the difference between IRIF and PpoI DSBs relates to the DSB chemistry or location.
We thank the reviewer for this important point. We agree that I-PpoI induces DNA breaks both within rDNA repeats and at additional genomic loci.
To address this, we have now analysed the formation of gH2AX-positive nucleolar caps and non-nucleolar gH2AX foci over time following I-PpoI expression (new Figure 1–figure supplement 2). We find that nucleolar caps form rapidly and are prominent at early time points, whereas gH2AX foci accumulate more gradually.
These results indicate that nucleolar caps and non-nucleolar DNA damage responses can be distinguished both spatially and temporally, and support the use of nucleolar caps as a specific readout for rDNA damage in our study.
In addition, we note that RAD51 recruitment to IR-induced foci is not affected by MDC1 loss, suggesting that the requirement for MDC1 in RAD51 loading is specific to nucleolar rDNA breaks rather than reflecting differences in DSB chemistry alone. We have clarified this point in the Discussion.
Additional points:
(5) Page 4 top: Shieldin.
Corrected.
(6) The general reader will be interested to learn about the connection of the Treacle function with Treacher Collins syndrome. Maybe a paragraph could be added to discuss this?
We thank the reviewer for this suggestion. We agree that the relationship between Treacle and Treacher Collins syndrome may be of interest to a broad readership. Since the developmental pathology of Treacher Collins syndrome is currently thought to arise primarily from impaired ribosome biogenesis and nucleolar dysfunction rather than defective nucleolar DNA damage signalling, we felt that an extensive discussion would be beyond the scope of the present study. We have, however, added a brief statement introducing Treacle as the product of the TCOF1 gene mutated in Treacher Collins syndrome and noting that whether its DNA damage response function contributes to disease pathology remains an open question.
(7) Figure 7: A short explanation could be added as to why hypoxia conditions were chosen for the p53-deficient cell lines.
We thank the reviewer for pointing this out. We have added a brief explanation in the figure legend to clarify that hypoxia conditions were used to stabilise replication stress and enhance detection of DNA repair intermediates in p53-deficient cells.
(8) A short statement on whether the repair of nuclear DBS is affected by Treacle could be added.
We thank the reviewer for this interesting point. While our study focuses on nucleolar DNA damage, we did not observe evidence that Treacle is required for the repair of non-nucleolar DSBs. We have added a brief statement in the Discussion to clarify that Treacle appears to function specifically in the nucleolar DNA damage response.
