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. 2025 Jan 2;85(1):78-90.e3.
doi: 10.1016/j.molcel.2024.11.006. Epub 2024 Dec 3.

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

Rajula Elango  1 , Namrata M Nilavar  1 , Andrew G Li  1 , Daniel Nguyen  1 , Emilie Rass  1 , Erin E Duffey  1 , Yuning Jiang  1 , Abdulkadir Abakir  2 , Nicholas A Willis  1 , Jonathan Houseley  2 , Ralph Scully  3
Affiliations

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

Rajula Elango et al. Mol Cell. .

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.

Keywords: BRCA1; Camptothecin; DNA nick; DNA-protein crosslink; Flp recombinase; Tus/Ter; break-induced replication; homologous recombination; replication fork breakage; replication stress.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

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