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Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. Significance: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype “BRCAness,” yielding promising biomarkers, targets, and opportunities to resensitize refractory disease. See related commentary by Canman, p. 1214

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress–inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

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SUMMARY The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.

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BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation.We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions. Schlacher et al., 2012). Consistent with the DSB inducing model of therapy response in BRCA deficient cells, restoration of HR or FP are associated with chemoresistance (Bouwman et al., 2010; Bunting et al., 2010; Chaudhuri et al., 2016; Edwards et al., 2008; Sakai et al., 2008). However, this model of therapy response is challenged by recent reports indicating that chemotoxic agents do not initially induce DSBs or even pause DNA replication forks (Huang et al., 2013; Mutreja et al., 2018; Zellweger et al., 2015). Indeed, PARPi accelerates DNA replication (Maya-Mendoza et al., 2018). To square this finding with the framework that HR and/or FP defects drive PARPi therapy response, it was proposed that high speed DNA replication ultimately induces DSBs (Maya-Mendoza et al., 2018; Quinet and Vindigni, 2018). However, we recently proposed a competing model in which ssDNA gaps underlie the BRCA deficiency phenotype, and not DSBs and we propose are fundamental to the mechanismof-action of genotoxic therapies (Panzarino et al., 2019). 2011;Here, we provide evidence that the genotoxic lesion driving PARPi synthetic lethality in BRCA deficient cancer is wide-spread single-stranded DNA (ssDNA) gaps. Gap induction is associated with PARPi potentially due to its role in the repair of ssDNA breaks, processing Okazaki fragments, or regulating replication-fork reversal -a mechanism by which replication forks reverse direction when confronted with replication obstacles (Berti et al. Sogo et al., 2002). Critically, we demonstrate that PARPi gaps are compounded in BRCA-deficient cells, but suppressed in cell lines and tumors with intrinsic, genetic or de novo mechanisms of PARPi resistance. These findings highlight that a key function of the BRCA-RAD51 proteins is to limit replication gaps (Hashimoto et al., 2010; Kolinjivadi et al., 2017a; Kolinjivadi et al., 2017b; Zellweger et al., 2015, Panzarino et al., 2019 and that loss of this function confers therapy response. RESULTS PARPi generates s...