The WRN helicase/exonuclease protein is required for proper replication fork recovery and maintenance of genome stability. However, whether the different catalytic activities of WRN cooperate to recover replication forks in vivo is unknown. Here, we show that, in response to replication perturbation induced by low doses of the TOP1 inhibitor camptothecin, loss of the WRN exonuclease resulted in enhanced degradation and ssDNA formation at nascent strands by the combined action of MRE11 and EXO1, as opposed to the limited processing of nascent strands performed by DNA2 in wild-type cells. Nascent strand degradation by MRE11/EXO1 took place downstream of RAD51 and affected the ability to resume replication, which correlated with slow replication rates in WRN exonuclease-deficient cells. In contrast, loss of the WRN helicase reduced exonucleolytic processing at nascent strands and led to severe genome instability. Our findings identify a novel role of the WRN exonuclease at perturbed forks, thus providing the first in vivo evidence for a distinct action of the two WRN enzymatic activities upon fork stalling and providing insights into the pathological mechanisms underlying the processing of perturbed forks.

Werner syndrome (WS) is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi Anemia (FA), a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2−/− cells, NSC 617145 acted synergistically with very low concentrations of Mitomycin C (MMC) to inhibit proliferation in a WRN-dependent manner, and induce double strand breaks (DSBs) and chromosomal abnormalities. Under these conditions, ATM activation and accumulation of DNA-PKcs pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2−/− cells exposed to NSC 617145 and MMC, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination (HR) at ICL-induced DSBs. Thus, when the FA pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of FA-deficient tumors by their sensitization to DNA cross-linking agents is discussed.