Two-ended recombination at a Flp-nickase-broken replication fork
Rajula Elango, Namrata M. Nilavar, Andrew G. Li, Daniel Nguyen, Emilie Rass, Erin E. Duffey, Yuning Jiang, Abdulkadir Abakir, Nicholas A. Willis, Jonathan Houseley, Ralph Scully
Abstract
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 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.