M.T. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to total genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to handle mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and may become exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity. Replication stress represents a major source of genome instability stemming from sluggish rates GGT1 of DNA synthesis, aberrant source firing and frequent stalling of replication forks1. Treatment with providers that interfere with DNA replication (for example, hydroxyurea, aphidicolin), as well as oncogene overexpression2 are known to result in replication stress. Eukaryotic cells are prone to low levels of replication stress during normal, unchallenged cell cycle conditions. For example, barriers to fork progression (for example, DNA inter-strand mix links, DNA/RNA hybrids, G-quadruplexes) obstruct replication and cause fork stalling. To circumvent this problem and total genome duplication, cells have developed mechanisms for stabilizing and/or restarting stalled forks, some of which are dependent on the tumour suppressor BRCA2. BRCA2 is definitely a multifaceted protein best known for its function in promoting assembly of RAD51 filaments during homologous recombination (HR) restoration. BRCA2 is also required during DNA replication to protect stalled replication forks against nucleolytic degradation3 and to restoration collapsed replication forks through RAD51-dependent restart reactions4. In addition, BRCA2 plays a role during mitosis where it regulates the metaphase to anaphase transition by sustaining spindle assembly checkpoint (SAC) via BubR1 acetylation5. Upon gene deletion, main cells succumb to spontaneous double strand break (DSB) build up and checkpoint activation, which channel cells into senescence and apoptosis6,7. Malignancy cells lacking BRCA2, however, acquire additional mutations, for example in tumour suppressor genes such as p53, which together with upregulation of error-prone DSB restoration pathways sustain replication and proliferation8. Consequently, tolerance of high levels of replication stress and endogenous DNA damage enable survival of BRCA2-deficient malignancy cells. In eukaryotic cells, replication fork progression requires MUS81, a structure-specific endonuclease that functions in complex with its evolutionarily conserved partners EME1 (in candida and human being cells) or MMS4 (in budding candida)9. MUS81-dependent nucleolytic cleavage promotes HR-dependent restart of stalled forks10,11,12. In mouse and human being cells, the negative effects of MUS81 inactivation on fork restart have been examined following replication stress induced with hydroxyurea11,13,14. In addition, MUS81 is required in aphidicolin-treated human being cells for common fragile site (CFS) replication14,15 and DNA synthesis during mitosis16. Replication fork stalling at genomic areas that are hard to replicate or contain endogenous DNA lesions is definitely a hallmark of BRCA2 deficiency. We therefore investigated the effect of MUS81 on DNA replication in cells lacking BRCA2. Our results demonstrate that loss of MUS81 causes increased replication stress and reduced survival in BRCA2-deficient cells. These cells progress into mitosis with incompletely replicated DNA, visualized as multiple chromosome interlinks in anaphase. Moreover, BRCA2-deficient cells rely on MUS81 to continue DNA synthesis during mitosis, the absence of which causes severe chromosome segregation problems and G1 arrest. We propose that in cells lacking BRCA2, MUS81-dependent nucleolytic cleavage removes DNA bridges caused by under-replicated DNA and provides a mechanism to total replication in mitosis. Results JZL195 Replication problems in cells lacking MUS81 and BRCA2 To determine whether MUS81 and BRCA2 cooperate during unchallenged DNA replication, we measured replication rates using DNA fibre assays in H1299 human being cells upon inactivation of these factors (Fig. 1a and Supplementary Table 1). Consistent with earlier reports, we found that MUS81 inhibition experienced JZL195 no effect on fork progression17, while BRCA2 abrogation using a doxycycline (DOX)-inducible shRNA significantly decreased replication rate18. Strikingly, MUS81 inhibition in BRCA2-deficient cells caused a further slowdown in replication fork progression. We further observed that replication fork slowdown could be rescued by ectopically expressing siRNA-resistant wild-type MUS81, but not a catalytically inactive version of MUS81 (D338A/D339A), indicating that MUS81 nuclease activity is required to sustain replication in BRCA2-deficient cells (Fig. 1a). Wild-type MUS81 was indicated ectopically at levels similar to the catalytically inactive MUS81 (Supplementary Fig. 1a). Related reduction in fork velocity was observed using U2OS cells, in which MUS81 and BRCA2 were depleted using siRNAs (Supplementary Fig. 1b and Supplementary Table 2). To determine the potential contribution of replication JZL195 fork stalling to this phenotype, we measured the ability of MUS81- and/or BRCA2-depleted cells to restart stalled forks (Supplementary Fig. 1c)..