Mechanism for inverted-repeat recombination induced by a replication fork barrier

Léa Marie,Lorraine S Symington

doi:10.1038/s41467-021-27443-w

Abstract

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

Highlights

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes
Replication stress, defined as a slowing down or complete arrest of DNA synthesis during chromosome replication, has emerged as a primary cause of genomic instability, a hallmark of cancer and other human disorders associated with genomic rearrangements[1,70,71]
We show that a Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats stimulates recombination mediated by a unique genetic pathway, distinct from spontaneous non-allelic HR (NAHR) or post-replicative repair

Summary

Introduction

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. Replication forks become stressed as a result of DNA lesions, spontaneous formation of secondary structures, RNA–DNA hybrids, protein–DNA complexes, activation of oncogenes, or depletion of nucleotides[1]. These obstacles to the progression of replication can cause forks to slow down, stall and collapse. Replication fork reversal has emerged as a central remodeling process in the recovery of replication in both eukaryotes and bacteria[5] This process allows stalled replication forks to reverse their progression through the unwinding and annealing of the two nascent strands concomitant with reannealing of the parental duplex DNA, resulting in the formation of a four-way-junction, sometimes called a chicken-foot structure. A significant factor underlying chromosome rearrangements is the abundance of repeated sequences in eukaryotic genomes

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