Rad51 paralogues Rad55–Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

Abstract

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements, and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyzes the signature reactions of recombination, homology search and DNA strand invasion 1,2. Eukaryotes also possess Rad51 paralogs, whose exact role in recombination remains to be defined 3. Here we show that the budding yeast Rad51 paralogs, the Rad55-Rad57 heterodimer, counteract the anti-recombination activity of the Srs2 helicase. Rad55-Rad57 associate with the Rad51-ssDNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51/Rad55-Rad57 co-filament resists disruption by the Srs2 anti-recombinase by blocking Srs2 translocation involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogs in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 anti-recombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogs, which are known to be involved in cancer predisposition and human disease.

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…Alternatively, checkpoint inactivation might affect the stability of the Rad51 nucleoprotein filament in S/G2. Rad55 which is implicated in stabilization of Rad51 on DNA is phosphorylated in a Mec1‐Rad53‐dependent manner (Bashkirov et al , 2000) and the presence of functional Rad55/57 was reported to dictate the requirement for Srs2 in cells with DNA damage (Liu et al , 2011). Therefore, in checkpoint‐deficient cells, the lack of Rad55 phosphorylation might decrease the stability of Rad51 on DNA and promote stochastic replacement of Rad51 with RPA, thereby alleviating the need for Srs2.…”

Regulation of Recombination and Genomic Maintenance

“…The budding yeast Srs2 helicase has set the paradigm for reversing HR by dissociating Rad51 from ssDNA, providing a satisfying mechanism for the antirecombination function of Srs2 (Aboussekhra et al 1989(Aboussekhra et al , 1992Schiestl et al 1990;Krejci et al 2003;Veaute et al 2003). The Rad51 paralog complex Rad55 -Rad57 was found to counteract Srs2-mediated disruption of the Rad51 -ssDNA filament (Liu et al 2011a), suggesting a dynamic balance between the forward and backward reactions that may additionally be modulated by PTMs. Several mammalian proteins have been implicated in dissociating RAD51 from ssDNA (Heyer et al 2010).…”

Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

“…Yeast Rad51 is assisted in this process by accessory factors that include two Rad51 paralogs, namely, Rad55 and Rad57, and the recently described ensemble of Psy3, Csm2, Shu1, and Shu2 (the PCSS complex) (Ball et al, 2009;Krejci et al, 2012;Sasanuma et al, 2013a;Shor et al, 2005;Tao et al, 2012;Qing et al, 2011). Srs2, a major anti-recombinase, is a 3´ to 5´ DNA helicase that interacts with and dissociates Rad51 from single-stranded DNA (ssDNA) in vitro (Antony et al, 2009;Liu et al, 2011). The Rad55-Rad57 heterodimer stabilizes Rad51 filaments to resist the disruption induced by Srs2 (Liu et al, 2011).…”

“…Srs2, a major anti-recombinase, is a 3´ to 5´ DNA helicase that interacts with and dissociates Rad51 from single-stranded DNA (ssDNA) in vitro (Antony et al, 2009;Liu et al, 2011). The Rad55-Rad57 heterodimer stabilizes Rad51 filaments to resist the disruption induced by Srs2 (Liu et al, 2011). Shu1 functions in Rad51-dependent homologous recombination, and the PCSS complex inhibits Srs2 to stabilize Rad51-ssDNA filaments (Mankouri et al, 2007).…”