Two-ended recombination at a Flp-nickase-broken replication fork

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. [Display omitted] •The Flp-nickase models camptothecin-induced TopI lesions in mammalian cells•BRCA1-mediated DNA end resection is dispensable for Flp-nick-induced HR•Collision of one replication fork with the Flp-nick induces two-ended HR•The replication fork can bypass some Flp-nicks, likely those on the lagging strand Replication fork collision with a nicked DNA template was proposed to generate exclusively one-ended breaks. Elango et al. show that collision of a single replication fork with a Flp-induced nick can stimulate two-ended homologous recombination. Thus, the replisome can bypass some Flp-nicks, leaving a two-ended break in its wake.