Treacle and MDC1 coordinate rDNA break repair by homologous recombination

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)

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.