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Replication fork collision with a DNA nick can generate a one-ended break, fostering genomic instability. The opposing fork's collision with the nick could form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. A Flp-nick induces two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR pathways induced by a replication-independent break and the Flp-nickase differ in their dependence on BRCA1, MRE11, and CtIP. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a Tus/Ter replication fork barrier (RFB). Flp-nickase-induced STGC remained robust and two ended. Thus, a single replication fork's collision with a Flp-nick triggers two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of nicked DNA.

The fork protection complex (FPC), composed of Mrc1, Tof1, and Csm3, supports rapid and stable DNA replication. Here, we show that FPC activity also introduces DNA damage by increasing DNA topological stress during replication. Mrc1 action increases DNA topological stress during plasmid replication, while Mrc1 or Tof1 activity causes replication stress and DNA damage within topologically constrained regions. We show that the recruitment of Top1 to the fork by Tof1 suppresses the DNA damage generated in these loci. While FPC activity introduces some DNA damage due to increased topological stress, the FPC is also necessary to prevent DNA damage in long replicons across the genome, indicating that the FPC is required for complete and faithful genome duplication. We conclude that FPC regulation must balance ensuring full genome duplication through rapid replication with minimizing the consequential DNA topological stress-induced DNA damage caused by rapid replication through constrained regions.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.