Homologous Recombination Restarts Blocked Replication Forks at the Expense of Genome Rearrangements by Template Exchange

Abstract

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)

“…Finally, we observed sensitivity to hydroxyurea (HU) in rad16-249 and rhp14Δ mutants, but again not in rad13Δ. HU causes fork stalling due to nucleotide depletion, and restart occurs via recombination-based mechanisms (Meister et al 2007;Lambert et al 2010;Sabatinos et al 2012).…”

“…Finally, we observed sensitivity to hydroxyurea (HU) in rad16-249 and rhp14Δ mutants, but again not in rad13Δ. HU causes fork stalling due to nucleotide depletion, and restart occurs via recombination-based mechanisms (Meister et al 2007;Lambert et al 2010;Sabatinos et al 2012).Slx4 and Saw1 are proposed to function as scaffolds that assemble XPF and other structure-specific endonucleases (Lyndaker and Alani 2009;Kashiyama et al 2013;Li et al 2013;Wan et al 2013). In contrast to budding yeast (Flott et al 2007;Li et al 2008), neither slx4Δ nor saw1Δ is sensitive to UV or MMS in fission yeast (Figure S3 and (Coulon et al 2006).…”

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…4,34,35 An alternative hypothesis, in which a DSB intermediate is not invoked, proposes that template switches mediate HR at the Tus/Ter block, as has been suggested from analysis of a site-specific replication fork barrier in Schizosaccharomyces pombe. 36 For the purpose of discussion, we assume a DSB model of Tus/Terinduced HR; however, the mechanisms discussed here could also apply to a template switch model. We propose that the LTGC bias noted in HR reporters containing an extended array of Ter sites is a consequence of increased spatial separation of the sites of stalling of the converging replication forks (Fig.…”

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…One interesting possibility is that the yeast replisome itself may physically destabilize (or actively trigger the localized degradation of) DNA-bound Tus following RF arrest. Importantly, because HRR is not required to restart RFs following pausing at Tus-Ter modules, our system can be distinguished from the RTS1 barrier previously characterized in S. pombe 8,37 . Rather, we propose that transient RF pausing at Tus-Ter modules may cause a site-specific single-stranded DNA (ssDNA) gap that is post-replicatively repaired by HRR.…”

“…HRR has been strongly implicated in the restart and repair of stalled RFs 36 . Indeed, the RTS1 RF barrier in S. pombe requires Rad22 (Rad52 S.c. ), Rad50 (Rad50 S.c. ) and Rhp51 (Rad51 S.c. ) for RF restart 8,37 . We therefore analysed the consequences of RF pausing ARTICLE at Tus-Ter modules in rad50, rad51 and rad52 mutants.…”