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Summary Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells’ ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1’s inhibition in EXD2−/− cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2’s nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2’s depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection., Graphical Abstract, Highlights • EXD2 is required for cell survival in response to replicative stress • EXD2 protects replication forks from over resection by counteracting fork reversal • EXD2 is synthetic lethal with the deficiency in BRCA1/2 genes, Nieminuszczy et al. identify a key function of the EXD2 nuclease in DNA replication and alternative end-joining. EXD2 localizes to replication forks and promotes their stabilization by counteracting fork regression. Loss of EXD2 results in sensitivity to replicative inhibitors, degradation of regressed forks, and compromises survival of BRCA1/2-deficient tumors.

The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single molecule technique for visualizing encounters of replication forks with ICLs, as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs, and that these lesions are not always absolute blocks to replication.