EXD2’s exonuclease activity facilitates the generation of ssDNA
The MRN complex processes DSBs to generate ssDNA, which requires MRE11’s 3′-5′ exonuclease activity 20, 35. Interestingly, EXD2 has a predicted exonuclease fold, which has sequence homology to the 3′-5′ exonuclease domain of the Werner syndrome protein (WRN). Analysis of the alignment between EXD2 and WRN identified two key amino acids (D108 and E110) within the putative exonuclease domain of EXD2, which are also highly conserved in other DnaQ type exonucleases, including WRN, that coordinate the binding of metal ions within the active site 36 (Supplementary Fig. 3a). Mutation of the equivalent residues in WRN (D82 and E84) renders the protein devoid of nuclease activity 37. Therefore, we hypothesized that the equivalent residues in EXD2 may be also required for its putative nuclease activity. To test this, we expressed the full length GST-tagged EXD2 and the D108A and E110A mutant protein in bacteria, and purified them to apparent homogeneity (Supplementary Fig. 3b). Next, we tested the activity of these purified proteins on single stranded DNA radiolabeled on the 3′ or 5′ end (Fig. 4a and b). We found that purified EXD2, but not the D108A and E110A mutant, exhibited a robust nuclease activity on short 5′ labeled ssDNA (Fig. 4a). Furthermore, a time course of the 3′ labelled substrate digestion indicates that EXD2 degrades the labelled DNA strand from the 3′ end, as evidenced by the release of the single labelled nucleotide (Fig. 4b). This data shows that EXD2 displays a 3′-5′ exonuclease activity in vitro. Moreover, under these conditions the WT protein exhibited only weak activity towards blunt end double stranded DNA (Fig. 4c). To verify this data, we also identified a highly-soluble truncated form of EXD2 (spanning residues lysine 76 through to valine 564, containing the predicted exonuclease domain) that can be produced at very high yields and purity in a three-step procedure (Supplementary Fig. 3c-e). This version of EXD2, and its D108A E110A variant, behaved indistinguishably from full-length EXD2 (Supplementary Fig. 3f). In addition, the protein showed only a weak activity towards dsDNA with resected 3’end (Fig. 4d), and did not display any endonuclease activity on ssDNA or dsDNA with biotin/streptavidin blocked 3′ end (Fig. 4e). Importantly, purified EXD2 displayed a robust exonuclease activity, which co-elutes with the protein (Fig. 4f). Thus our data identify EXD2 as a bone fide exonuclease with a 3′-5′ polarity.
To address the potential biological significance of EXD2’s exonuclease activity, we tested whether this activity was required to promote DNA-end resection in vivo. To this end, we examined the phenotypes of two independently derived U2OS clones stably expressing wild-type or the nuclease-dead (D108A and E110A) EXD2 mutant. The endogenous protein was depleted with siRNA targeting the 3′ un-translated region (UTR) of EXD2 (Supplementary Fig. 4a). Notably, cells expressing the nuclease-dead protein did not correct phenotypes associated with EXD2 deficiency as compared to cells expressing wild-type EXD2 (Fig. 5a-d). This result is consistent with our data showing a requirement for EXD2 in the processing of DSBs into ssDNA. Cells overexpressing WT EXD2 displayed elevated resection. In this regard we note that overexpression of WT MRE11 also increases resection efficiency resulting in elevated levels of RPA foci formation and RPA phosphorylation 38.