Shu Proteins Promote the Formation of Homologous Recombination Intermediates That Are Processed by Sgs1-Rmi1-Top3

Abstract

CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination repair (HRR), their precise role(s) within this pathway remains poorly understood. Here, we have identified a specific role for the Shu proteins in a Rad51/Rad54-dependent HRR pathway(s) to repair MMS-induced lesions during S-phase. We show that, although mutation of RAD51 or RAD54 prevented the formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex. INTRODUCTIONHomologous recombination repair (HRR) is a well-conserved cellular process for the repair of single-strand DNA (ssDNA) gaps and double-strand DNA breaks (DSBs) that can arise during DNA synthesis or as a result of replication fork stalling during S-phase. Although many of the key proteins involved in HRR have been identified (reviewed in Paques and Haber, 1999;Sung et al., 2003;West, 2003;Krogh and Symington, 2004), in some cases their precise function(s) remains to be identified. Moreover, the mechanisms for suppression of inappropriate HRR in S-phase are only poorly defined. It is likely that HRR must be carefully regulated and/or executed in dividing cells, as inappropriate or excessive HR can lead to genome rearrangements and cancer in mammals. This is exemplified by the cancer-predisposition disorder, Bloom's syndrome, which is caused by mutation in the human BLM gene (reviewed in German, 1993). Because the BLM protein, in conjunction with its associated proteins, hTOPOIII␣ and hRMI1 (Johnson et al., 2000;Wu et al., 2000;Yin et al., 2005), can catalyze dissolution of HRR intermediates in vitro Plank et al., 2006;Raynard et al., 2006;Wu et al., 2006), it is likely that unprocessed and/or aberrantly processed HRR intermediates at least partly contribute to the cellular defects in Bloom's syndrome. Indeed, Bloom's syndrome cells classically demonstrate elevated levels of sister chromatid exchanges, mitotic recombination, and genome instability. Mutation of the BLM, hTOPOIII␣, or hRMI1 homologues in Saccharomyces cerevisiae (SGS1, TOP3, or RMI1, respectively) similarly causes sensitivity to genotoxic agents, hyper-recombination, and synthetic lethality with mutations in other genes also implicated in HRR (e.g., MUS81 and SRS2; Gangloff et al., 1994;Watt et al., 1996;Gangloff et al., 2000;Mullen et al., 2001;Fabre et al., 2002;Chang et al., 2005;Mullen et al., 2005). Furthermore, unresolved HRR intermediates have been directly visualized by two-dimensional (2D) gel electrophoresis in cells lacking Sgs1 or in cells w...

Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

“…1B). Cell samples from each time point were treated with psoralen and then exposed to UV light to produce cross-linked DNA interstrands, which are joint molecules (i.e., SEIs and dHJs) ( Absence of Shu1 delays meiotic recombination and reduces crossover recombinants Psy3, Csm2, Shu1, and Shu2 (hereafter referred to as the "PCSS complex") were originally identified from a genetic screen as mutational suppressors of the top3Δ or sgs1Δ growth defect in Saccharomyces cerevisiae (Mankouri et al, 2007;Shor et al, 2005). The proteins of the PCSS complex physically interact and fall in the same epistasis group (Ito et al, 2001;Shor et al, 2005).…”

“…The proteins of the PCSS complex physically interact and fall in the same epistasis group (Ito et al, 2001;Shor et al, 2005). Furthermore, these single mutants show similar levels of sensitivity to methylmethane sulfonate (MMS) (Mankouri et al, 2007), implying that the PCSS complex components play together in vivo in the same pathway. To observe the conditional effect of Shu1, we constructed a copper-specific conditional expression allele by replacing the Shu1 promoter with the CUP1 promoter, which is repressed in the absence of copper ( Fig.…”

“…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). In the most recent study, core Psy3-Csm2 dimer was observed to demonstrate structural similarity to Rad51/RecA (Sasanuma et al, 2013a).…”

“…In the most recent study, core Psy3-Csm2 dimer was observed to demonstrate structural similarity to Rad51/RecA (Sasanuma et al, 2013a). Furthermore, because Shu2 and Srs2 physically interact with each other (Ito et al, 2001;Martín et al, 2006), it is possible that the PCSS complex promotes strand invasion by coordination with Rad51 to prevent Srs2 from disrupting Rad51 nucleofilaments (Mankouri et al, 2007). Alternatively, Psy3-Csm2, which is a component of the PCSS complex, could directly interact with ssDNA and stabilize Rad51 nucleoprotein filaments (Sasanuma et al, 2013a).…”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…In particular, Sgs1 and Top3 are suggested to resolve recombination structures originating from sister chromatid junctions at damaged replication forks, while Srs2 prevents their formation (Liberi et al, 2005;Mankouri and Hickson, 2006;Mankouri et al, 2007). mph1 mutants are not deficient in HR, as determination of spontaneous mitotic recombination rates demonstrated.…”