Distinct Roles of Mus81, Yen1, Slx1-Slx4, and Rad1 Nucleases in the Repair of Replication-Born Double-Strand Breaks by Sister Chromatid Exchange

Centro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad de Sevilla-CSIC, Seville, Spain.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/2012; 32(9):1592-603. DOI: 10.1128/MCB.00111-12
Source: PubMed


Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

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    • "The failure to detect a decrease in chromosomal Ade+ recombinants in the absence of Mus81 could be due to Yen1 cleavage of the BIR intermediate (Blanco et al. 2010; Ho et al. 2010; Agmon et al. 2011; Muñoz-Galván et al. 2012). Because the mus81Δ yen1Δ diploid grows very slowly and has low transformation efficiency we were unable to generate sufficient Ura+ Ade+ transformants for meaningful analysis. "
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    ABSTRACT: Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1 and Yen1 structure-selective nucleases act redundantly to facilitate BIR.
    Genetics 02/2014; 196(4). DOI:10.1534/genetics.114.162297 · 5.96 Impact Factor
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    • "In aggregate, these results demonstrate that the vast majority of DSBs produced following replication fork stalling in ATR-deficient cells occurs in an RNF4-dependent manner and that PLK1 appears to play a secondary or compensatory role in this process. The SLX4–endonuclease complex has previously been implicated as a mediator of replication fork collapse, particularly in cooperation with the MUS81–EME1 complex (Froget et al. 2008; Forment et al. 2011; Matos et al. 2011; Gallo-Fernandez et al. 2012; Munoz-Galvan et al. 2012; Schwartz et al. 2012; Szakal and Branzei 2013). To examine the involvement of this complex in the DSBs generated upon fork stalling in ATR-deficient cells, Crelox conditional SLX4 cells were used to delete Slx4 and determine the effect of its absence on DSB generation following ATR inhibition and fork stalling. "
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    ABSTRACT: The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.
    Genes & development 10/2013; 27(20):2259-73. DOI:10.1101/gad.223180.113 · 10.80 Impact Factor
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    • "The mph1D yen1D double mutant showed no reduction in PE and the same profile of recombinants as the mph1D single mutant, suggesting that Yen1 does not contribute appreciably to CO formation in the absence of Mph1. The severe growth defect of the mph1D mus81D yen1D triple mutant suggests that Yen1 might be required to alleviate the accumulation of some toxic intermediates in the absence of Mus81-Mms4 and Mph1 and is consistent with Yen1 overlapping to some extent with Mus81-Mms4 in the formation of COs (Agmon et al., 2011; Ho et al., 2010; Mazó n et al., 2012; Muñ oz-Galvá n et al., 2012). The triple mutant yielded a low number of red/whitesectored recombinants and a PE of 47% (Figures 5 and S5A), but in this limited pool the CO fraction was reduced to 2.8% and BIR increased to 13.8%. "
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    ABSTRACT: Homology-dependent repair of double-strand breaks (DSBs) from nonsister templates has the potential to generate loss of heterozygosity or genome rearrangements. Here we show that the Saccharomyces cerevisiae Mph1 helicase prevents crossovers between ectopic sequences by removing substrates for Mus81-Mms4 or Rad1-Rad10 cleavage. A role for Yen1 is only apparent in the absence of Mus81. Cells lacking Mph1 and the three nucleases are highly defective in the repair of a single DSB, suggesting that the recombination intermediates that accumulate cannot be processed by the Sgs1-Top3-Rmi1 complex (STR). Consistent with this hypothesis, ectopic joint molecules (JMs) accumulate transiently in the mph1Δ mutant and persistently when Mus81 is eliminated. Furthermore, the ectopic JMs formed in the mus81Δ mutant contain a single Holliday junction (HJ) explaining why STR is unable to process them. We suggest that Mph1 and Mus81-Mms4 recognize an early strand exchange intermediate and direct repair to noncrossover or crossover outcomes, respectively.
    Molecular cell 10/2013; 52(1):63-74. DOI:10.1016/j.molcel.2013.09.007 · 14.02 Impact Factor
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