EXD2 cooperates with MRE11 in the repair of DSBs
The in vivo resection that initiates DSB repair is catalysed by the MRN complex 6, 7, 14, 39. To test whether or not EXD2 collaborates in this process with MRE11 we analysed the kinetics of RPA foci formation (a marker of resection) in cells depleted for either of these proteins or concomitantly depleted for both EXD2 and MRE11. We found that combined depletion resulted in a comparable inhibition of resection as observed for depletion of MRE11 alone (Fig. 6a). A similar relationship was also observed for RAD51 foci (Supplementary Fig. 4b and c). Interestingly, the defect observed in EXD2-depleted cells was slightly weaker than that observed in MRE11 alone, suggesting that MRE11 functions upstream of EXD2 in DNA resection, perhaps initiating resection through its endonuclease activity. Indeed, it has been suggested recently that MRE11 may create multiple nicks on the strand being resected that could serve as additional exonuclease entry sites to further enhance nucleolytic processing 21, 22. We tested this notion in several ways. First, we analysed if MRN-dependent DNA resection is accelerated in the presence of EXD2. Purified EXD2 and the MRN complex (MRE11, RAD50, NBS1) were incubated together with circular single-stranded PhiX174 DNA. This substrate requires initial endonuclease-dependent nicking by the MRN complex in order to undergo resection. As previously reported, the MRN complex exhibited nuclease activity under these conditions 16 (Fig. 6b). Importantly, combining EXD2 with the MRN complex resulted in increased ssDNA degradation in vitro (Fig. 6b and c). As expected, addition of exonuclease-dead EXD2 protein to the MRN complex resulted in DNA degradation similar to what we observed for MRN alone (Fig. 6b and c). Secondly, we predicted that EXD2 should be able to initiate resection from a nicked and a gapped duplex substrate designed to mimic the substrates generated by MRE11 endonuclease activity during the initial stage of DNA-end resection. Strikingly, EXD2 exhibited robust exonuclease activity on both the nicked and gapped substrates (Fig. 6d, e and f and Supplementary Fig. 4d and e). Taken together, these data show that EXD2 functionally collaborate with the MRN complex in promoting DNA degradation.
To gain more functional insight into the role of EXD2’s exonuclease activity in DNA-end resection in vivo, we took advantage of the recently developed small molecule inhibitors targeting the exo- or endonuclease activity of MRE11 23. Inhibition of MRE11 endonuclease activity resulted in almost total inhibition of resection, whereas cells treated with the MRE11 exonuclease inhibitor showed milder resection defect (Fig. 7a) as reported previously 23. Knockdown of EXD2 alone resulted in a resection defect significantly stronger (p<0.0001) than the one observed in cells treated with the MRE11 exonuclease inhibitor alone. Depletion of EXD2 in the presence of the MRE11 exonuclease inhibitor did not decrease efficiency of resection further than what was achieved with EXD2 depletion alone (Fig. 7a). Interestingly, some residual resection was still observed in cells concomitantly depleted for EXD2 and incubated with the MRE11 exonuclease inhibitor, indicating either an involvement of another exonuclease in this step of DNA-end processing 23 or that EXD2 knockdown or MRE11 exonuclease inhibition was not complete. Nevertheless, this data suggests that both the exonuclease activity of EXD2 and that of MRE11 function within the same pathway and are required for efficient DNA end-resection. Knockdown of EXD2 in cells treated with an inhibitor targeting MRE11’s endonuclease activity showed a similar resection defect when compared to the inhibitor alone (Fig. 7a), supporting the notion that EXD2 functions downstream of MRE11’s endonuclease activity.
Next, we asked which part of the resection process i.e. short-range vs. long-range resection is affected in cells depleted for EXD2. To test this, we depleted EXD2 in a cell line allowing for the induction of a DSB in a specific genomic locus in vivo 40. Then we analysed the efficiency of DNA resection at this DSB by q-PCR at two positions: one located close to the break (335bp downstream of the break - short range resection) and the other located at 1618bp from the break (long range resection) 40. Interestingly, we found that EXD2-depletion affected both short range as well as long-range resection (Fig. 7b and c and Supplementary Fig. 4f). Knockdown of MRE11 resulted in a similar albeit stronger resection phenotype thus providing further evidence to support its upstream function in this process. Importantly, concomitant depletion of both proteins did not further potentiate the resection defect.
Given that both EXD2 and MRE11 regulate DSB resection, we tested the effect of their combined depletion on homologous recombination using the DR-GFP assay. In support of their role in this process, we observed that combined depletion of EXD2 and MRE11 did not decrease homologous recombination efficiency further than what we observed in the single knockdowns (Fig. 6d and Supplementary Fig. 4g).
These findings therefore show that EXD2 promotes DNA end resection and homologous recombination by enhancing the generation of ssDNA through a common mechanism with the MRN complex. Furthermore, it seems likely that MRE11 functions upstream of EXD2 in this process, likely initiating resection through its endonuclease activity.
To verify and extend the above conclusions, we used CRISPR-Cas9 nickase based gene editing 41 in HeLa cells to generate EXD2−/− clones (Supplementary Fig. 5a). The use of Cas9 nickase has been recently shown to minimize any off-target effects 42. Comparable to siRNA treated U2OS cells, EXD2−/− HeLa cells showed dramatically decreased RPA focus formation in response to CPT (Supplementary Fig. 5b and c), diminished RPA2 phosphorylation on S4/S8 (Supplementary Fig. 5d) and decreased survival in response to CPT (Supplementary Fig. 5e). We also tested if EXD2 depletion affects the MRE11 or CtIP protein stability and/or their recruitment to DSBs. We found this not to be the case, as cells lacking EXD2 had similar level of endogenous MRE11 or CtIP as the WT control (Supplementary Fig. 5f). Likewise, MRE11 or GFP-CtIP localization to DSBs induced by microirradiation 43 was not affected (Supplementary Fig. 6a, b and c). These finding therefore establish EXD2 as an important regulator of DSBs resection.