Mus81-Mms4 Functions as a Single Heterodimer To Cleave Nicked Intermediates in Recombinational DNA Repair

Abstract

The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3= flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3= flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions. Robin Holliday first proposed a mechanism for crossover formation (29). Based on fungal tetrad data, he envisioned that nick-induced heteroduplex formation could result in a DNA intermediate composed of four intact strands after ligation of the strand interruptions. This intermediate was later termed the Holliday junction (HJ). Cleavage across the two alternative planes of this junction would result in crossover (CO) or noncrossover (NCO) products, depending on the orientation of cleavage. Biochemical analysis of the bacterial RuvC nuclease supports this model and provides a paradigm for a class of enzymes called Holliday junction resolvases. These nucleases form homodimeric complexes to deliver two coordinated and symmetric endonucleolytic cuts that generate DNA ends that can be directly ligated to form recombinant products (reviewed in reference 38). However, RuvC is not evolutionarily conserved in eukaryotes, and the specific mechanisms of crossover formation in eukaryotes are still undefined. Refinement and expansion of the original Holliday model have produced the current model of double-strand break repair, in which one subpathway is defined by the formation of a double Holliday junction (dHJ) (46). Physical analysis has demonstrated the existence of dHJs in meiotic and mitotic recombination (12, 48). However, these studies could not establish whether these junctions were truly dHJs, i.e., with each individual junction having four uninterrupted strands, or were nicked junctions in a dHJ population where each strand could be found to be full length (for more discussion, see reference 49). Hence, the importance of Holliday junctions...

FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress

“…In relation to a stalled fork, this would allow unwinding of the lagging strand, potentially generating a substrate for MUS81. However, the precise substrate recognized by MUS81 at the fork is not known, but possibilities include Holliday junctions generated by replication fork regression or the stalled fork itself 8,21,22 . Our data on the role of MUS81 in the response to HU are in agreement with Hanada et al 6 with regard to MUS81-induced DNA DSB formation.…”

Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

“…S2). Perhaps our inability to cytologically detect MUS81 is related to the observation that Mus81-Mms4 acts as a single heterodimer (25), whereas Mlh1 appears to have a structural role also (26). Regardless, in the absence of an effective antibody to MUS81 protein, we classified and mapped RNs from nuclei in which each of the twelve chromosomes had at least one MLH1 focus to minimize the chance that an RN was not labeled with MLH1 antibodies for technical reasons (see SI Appendix, SI Materials and Methods for details and tests of selection criteria).…”

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

“…PLK1 and orthologs in yeast (S. cerevisiae Cdc5) have also been shown to regulate the activation of MUS81-EME1 and stimulate its association with the SLX4-endonuclease complex (Matos et al 2011;Gallo-Fernandez et al 2012;Munoz-Galvan et al 2012;Schwartz et al 2012;Szakal and Branzei 2013). Although it is not clearly essential for fork collapse in all cell types, the Mus81-Eme1 complex in Schizosaccharomyces pombe plays a crucial role in DSB generation at stalled replication forks (Froget et al 2008), and a similar relationship has been observed in mammalian cells when CHK1 is inhibited (Forment et al 2011).…”