Error-free DNA replication is paramount to maintaining genomic integrity. Despite being highly regulated, the process of DNA replication is often challenged by various intrinsic and extrinsic sources of replication stress. Failure to maintain the DNA replication quality reduces genomic stability, cell survival and results in diseases, such as cancer. Thus, cells rely on the replication stress response that detects perturbations in DNA replication and pauses or arrests cellular replication. Similar to other intrinsic replication obstacles, oncogene expression also induces the replication stress response that acts as a barrier to cancer, thereby mystifying how cancer develops. Here, we demonstrate that oncogene expression, similar to other replication stress inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness unless counteracted by translesion synthesis (TLS). Moreover, we find that TLS subverts the replication stress response in a wide range of cancer cell lines indicating that TLS is a previously unappreciated and unique cancer vulnerability. Mechanistically, we reveal that upon replication stress, TLS restricts replication fork slowing, reversal, and fork degradation, while maintaining continuous replication. Furthermore, 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 other therapies that induce replication gaps. Thus, our study places TLS at the center of cancer cell fitness as a necessary adaptation to overcome replication stress.

Mutations in the hereditary breast/ovarian cancer genes BRCA1/2 were shown to be synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). This toxicity is assumed to derive from PARPi-induced DNA double strand breaks (DSBs) that necessitate BRCA function in homologous recombination (HR) and/or fork protection (FP). However, PARPi accelerates replication forks. While high-speed replication could cause DSBs, the finding that PARPi leads to single-stranded DNA (ssDNA) gaps/nicks suggests replication gaps could also or alone be the cause of synthetic lethality. Here, we demonstrate that PARPi toxicity derives from replication gaps. Isogenic cells deficient in BRCA1 or the BRCA1-associated FANCJ, with common DNA repair defects in HR and FP, exhibit opposite responses to PARPi. Deficiency in FANCJ, a helicase also mutated in hereditary breast/ovarian cancer and Fanconi anemia, causes aberrant accumulation of fork remodeling factor HLTF and limits unrestrained DNA synthesis with ssDNA gaps. Thus, we predict replication gaps as a distinguishing factor and further uncouple HR, FP and fork speed from PARPi response. BRCA-deficient cells display excessive gaps that are diminished upon resistance, restored upon re-sensitization and when targeted augment synthetic lethality with PARPi. Furthermore, we define the source of gaps to defects in Okazaki fragment processing (OFP). Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1 but aberrantly low XRCC1 indicating a defective backup OFP pathway. Remarkably, 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. Collectively, our study highlights unprotected lagging strand gaps as a determinant of synthetic lethality, providing a new paradigm and biomarker for PARPi toxicity.