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Updated: Jun 5, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Published on: September 11, 2022
Two-ended recombination at a Flp-nickase-broken replication fork
Rajula Elango1, Namrata M Nilavar1, Andrew G Li1
1Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
None:
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.
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