Recent studies have uncovered an important role of protein ubiquitylation in the cellular responses to DNA damage. Specifically, RNF8-dependent amplification of damage signals at the proximity of DNA lesions is crucial for proper activation of the cell cycle checkpoint and maintenance of genome stability. Using 53BP1 and FK2 IRIF as markers, we provide strong evidence to support that UBC13 harbors the enzymatic activity that facilitates the RNF8-mediated checkpoint protein assembly. This is accomplished in part by the RNF8 FHA domain, which enables the specific loading of UBC13 onto DNA lesions. Moreover, we showed that the RNF8 RING domain is essential for bringing UBC13 to the proximity of sites of DNA breaks. Given the exquisite coordination of the RNF8-UBC13 complex and its instrumental role in damage signal amplification, it would be interesting to examine whether dysregulation of such complex formation might contribute to tumorigenesis.
UBC13 plays diverse roles in biological processes through its ability to interact with multiple substrate specificity-conferring E3 ubiquitin ligases. Intriguingly, we found that only a subset of RING domains derived from these E3 ubiquitin ligases was competent in anchoring and facilitating UBC13 function at DSBs. Given that UBC13 forms stable heterodimers with E2 variants MMS2 and UEV1A, one might speculate that these RING domains might structurally conform to target a specific heterodimer for function. Although it is possible that an unidentified E2 variant might exist and only transiently associate with UBC13 in response to DNA damage, the fact that both MMS2 and UEV1A are dispensable for the UBC13-mediated checkpoint protein assembly, together with the fact that the binding of UBC13 to E2 variants is not absolutely required for its function, suggests that UBC13 might function independently of E2 variants.
Our observation that UBC13 at DSBs is required for checkpoint protein assembly (see Fig. S6A and B in the supplemental material), which in turn is required for optimal cellular response to DNA damage, is also illustrated by the prolonged accumulation of pH2AX in UBC13−/− cells (see Fig. S6C in the supplemental material). Although MMS2 and UEV1A are dispensable for UBC13 function in DNA damage signaling, depletion of MMS2 also led to sustained basal DNA damage (see Fig. S7A, B, and C in the supplemental material [as indicated by the elevated percentages of 53BP1 and FK2 IRIF and pH2AX-positive cells in untreated cells]) and manifested prolonged recovery upon IR. Since MMS2 is pivotal for postreplication repair, these MMS2-associated deficits might reflect a failure of proper DNA repair in these cells.
Recent evidence suggests that UBC13 acts in concert with RNF8 in promoting checkpoint protein assembly. Our studies using a chimeric fusion protein gene that encodes the RNF8 FHA and UBC13 and was sufficient to restore RNF8 function in the DNA damage response are entirely consistent with this notion. It remains possible, however, that an unidentified E3 ubiquitin ligase may participate in substrate ubiquitylation at DSBs. Similar to RNF8, the E3 ubiquitin ligase RAD18 has been shown to promote DSB repair (1
). However, unlike RNF8, RAD18 is not required for FK2 and 53BP1 IRIF (unpublished observations and see Fig. S8A and B in the supplemental material).
In summary, we have provided strong evidence that the UBC13 gene encodes the enzymatic module which is essential for the RNF8-dependent propagation of DNA damage signals. We propose that the RNF8 RING domain selects a subset of UBC13 in vivo that enables substrate ubiquitylation that differs from those catalyzed by UBC13-MMS2 and UBC13-UEV1A heterodimers. Ubiquitylation events at DSBs subsequently allow sustained accumulation of checkpoint proteins, which in turn is essential for proper cellular response to genotoxic stress.