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

Abstract

SummaryCollision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, 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. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence onBRCA1. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a site-specific Tus/Terreplication fork barrier. Flp-nickase-induced STGC remained robust and two-ended. Thus, collision of a single replication fork with a Flp-nick can trigger two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of a nicked DNA template.

Strand asymmetry in the repair of replication dependent double-strand breaks

“…5 The recombination products generated by Flp-mediated nicking in mouse cells are also consistent with repair of a replication-dependent deDSB. 10 Surprisingly, we observe a deDSB with one of the gRNAs that targets the leading strand. We note that deDSBs are also detected using the HO-nick and Flp-nick systems in a plasmid context in budding yeast, and in the S. pombe genome using gpII and nCas9 nickases.…”

“…Although we did not identify products consistent with LTGC/BIR, studies in mammalian cells have shown an increased LTGC/STGC ratio in response to nCas9 or Flp-nick expression. 10,16,19 These findings could be explained by BIR initiating at a seDSB or to more extensive DNA synthesis associated with HR during S-phase in mammalian cells.…”

“…86 In agreement with our findings, nCas9 and Flp-nick-induced HR in murine and human cells is not suppressed by canonical NHEJ factors in contrast to Cas9-induced events. 10,17 Rare end-joined products can be detected in response to nCas9 expression in mammalian cells and these exhibit deletions with microhomologies at the junctions, a hallmark of TMEJ. Interestingly, end-joined products are increased in the absence of BRCA1, consistent with use of alternate error-prone repair pathways in the absence of HR.…”

“…In a recent study, the Flp-nick system was integrated into the genome of mouse cells and shown to stimulate RAD51-dependent recombination between closely linked repeats. 10 A minimal HO endonuclease recognition site that is mostly nicked by HO has been used to study the repair of replication-associated DSBs in a plasmid context. 11 In the plasmid system, the nick in converted to a double-ended DSB (deDSB) during replication; however, it is unclear whether this is due to fork convergence within the small replicon or to bypass of the lesion by the replicative helicase (CMG).…”

DNA Nicks Drive Massive Expansions of (GAA)nRepeats

“…A DNA nick in the lagging strand template can either lead to a fork collapse 116 or be bypassed by the replisome, leaving behind a two-ended DSB 117 , which can be repaired by sisterchromatid exchange 118 . A DNA nick in the leading strand template, in contrast, cannot be bypassed because the CMG helicase moving along the leading strand template would simply slide off 116,[119][120][121] , resulting in fork collapse and the formation of a one-ended DSB with no sister chromatid to rescue.…”