RNA interference inhibition of Mus81 reduces mitotic recombination in human cells

Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.     

Single Holliday junctions are intermediates of meiotic recombination

Crossing-over between homologous chromosomes facilitates their accurate segregation at the first division of meiosis. Current models for crossing-over invoke an intermediate in which homologs are connected by two crossed-strand structures called Holliday junctions. Such double Holliday junctions are a prominent intermediate in Saccharomyces cerevisiae meiosis, where they form preferentially between homologs rather than between sister chromatids. In sharp contrast, we find that single Holliday junctions are the predominant intermediate in Schizosaccharomyces pombe meiosis. Furthermore, these single Holliday junctions arise preferentially between sister chromatids rather than between homologs. We show that Mus81 is required for Holliday junction resolution, providing further in vivo evidence that the structure-specific endonuclease Mus81-Eme1 is a Holliday junction resolvase. To reconcile these observations, we present a unifying recombination model applicable for both meiosis and mitosis in which single Holliday junctions arise from single- or double-strand breaks, lesions postulated by previous models to initiate recombination.     

Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion

Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells.     

Mus81-Eme1-dependent and -independent crossovers form in mitotic cells during double-strand break repair in Schizosaccharomyces pombe

During meiosis, double-strand breaks (DSBs) lead to crossovers, thought to arise from the resolution of double Holliday junctions (HJs) by an HJ resolvase. In Schizosaccharomyces pombe, meiotic crossovers are produced primarily through a mechanism requiring the Mus81-Eme1 endonuclease complex. Less is known about the processes that produces crossovers during the repair of DSBs in mitotic cells. We employed an inducible DSB system to determine the role of Rqh1-Top3 and Mus81-Eme1 in mitotic DSB repair and crossover formation in S. pombe. In agreement with the meiotic data, crossovers are suppressed in cells lacking Mus81-Eme1. And relative to the wild type, rqh1Delta cells show a fourfold increase in crossover frequency. This suppression of crossover formation by Rqh1 is dependent on its helicase activity. We found that the synthetic lethality of cells lacking both Rqh1 and Eme1 is suppressed by loss of swi5(+), which allowed us to show that the excess crossovers formed in an rqh1Delta background are independent of Mus81-Eme1. This result suggests that a second process for crossover formation exists in S. pombe and is consistent with our finding that deletion of swi5(+) restored meiotic crossovers in eme1Delta cells. Evidence suggesting that Rqh1 also acts downstream of Swi5 in crossover formation was uncovered in these studies. Our results suggest that during Rhp51-dependent repair of DSBs, Rqh1-Top3 suppresses crossovers in the Rhp57-dependent pathway while Mus81-Eme1 and possibly Rqh1 promote crossovers in the Swi5-dependent pathway.     

Recombination: Holliday junction resolution and crossover formation

The heterodimeric nuclease Mus81-Eme1 has been proposed to be a Holliday junction resolvase and has now been found to be responsible for nearly all meiotic crossovers in fission yeast. The intriguing substrate preference of this enzyme for nicked Holliday junctions opens the possibility that crossover formation may not always involve double Holliday junctions.     

Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair

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.     

Meiotic DNA joint molecule resolution depends on Nse5�CNse6 of the Smc5�CSmc6 holocomplex

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81–Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81–Eme1 is unclear. In cells lacking Nse5–Nse6 of the Smc5–Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5–Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5–Smc6 complex in HJ resolution via Mus81–Eme1.     

Human Mus81-associated endonuclease cleaves Holliday junctions in vitro.

Mus81, a protein with homology to the XPF subunit of the ERCC1-XPF endonuclease, is important for replicational stress tolerance in both budding and fission yeast. Human Mus81 has associated endonuclease activity against structure-specific oligonucleotide substrates, including synthetic Holliday junctions. Mus81-associated endonuclease resolves Holliday junctions into linear duplexes by cutting across the junction exclusively on strands of like polarity. In addition, Mus81 protein abundance increases in cells following exposure to agents that block DNA replication. Taken together, these findings suggest a role for Mus81 in resolving Holliday junctions that arise when DNA replication is blocked by damage or by nucleotide depletion. Mus81 is not related by sequence to previously characterized Holliday junction resolving enzymes, and it has distinct enzymatic properties that suggest it uses a novel enzymatic strategy to cleave Holliday junctions.     

Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis

The double Holliday junction (dHJ) is generally regarded to be a key intermediate of meiotic recombination, whose resolution is critical for the formation of crossover recombinants. In fission yeast, the Mus81-Eme1 endonuclease has been implicated in resolving dHJs. Consistent with this role, we show that Mus81-Eme1 is required for generating meiotic crossovers. However, purified Mus81-Eme1 prefers to cleave junctions that mimic those formed during the transition from double-strand break to dHJ. Crucially, these junctions are cleaved by Mus81-Eme1 in precisely the right orientation to guarantee the formation of a crossover every time. These data demonstrate how crossovers could arise without forming or resolving dHJs using an enzyme that is widely conserved amongst eukaryotes.     

Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks

The processing of stalled replication forks and the repair of collapsed replication forks are essential functions in all organisms. In fission yeast DNA junctions at stalled replication forks appear to be processed by either the Rqh1 DNA helicase or Mus81-Eme1 endonuclease. Accordingly, we show that the hypersensitivity to agents that cause replication fork stalling of mus81, eme1, and rqh1 mutants is suppressed by a Holliday junction resolvase (RusA), as is the synthetic lethality of a mus81(-) rqh1(-) double mutant. Recombinant Mus81-Eme1, purified from Escherichia coli, readily cleaves replication fork structures but cleaves synthetic Holliday junctions relatively poorly in vitro. From these data we propose that Mus81-Eme1 can process stalled replication forks before they have regressed to form a Holliday junction. We also implicate Mus81-Eme1 and Rqh1 in the repair of collapsed replication forks. Here Mus81-Eme1 and Rqh1 seem to function on different substrates because RusA can substitute for Mus81-Eme1 but not Rqh1.     

Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities

Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks. Here we show that human cell-free extracts possess two distinct endonucleases that can cleave Holliday junctions. The first cleaves Holliday junctions in a structure- and sequence-specific manner, and associates with an ATP-dependent branch migration activity. Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome. Like RuvC-mediated resolution, the products can be religated. The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non-ligatable. Each nuclease has a defined substrate specificity: the branch migration-associated resolvase is highly specific for Holliday junctions, whereas the Mus81-associated endonuclease is one order of magnitude more active upon replication fork and 3'-flap structures. Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks. However, the Mus81-associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures.     

Mus81 cleavage of Holliday junctions: a failsafe for processing meiotic recombination intermediates?

The Holliday junction (HJ) is a central intermediate of homologous recombination. Its cleavage is critical for the formation of crossover recombinants during meiosis, which in turn helps to establish chiasmata and promote genetic diversity. Enzymes that cleave HJs, called HJ resolvases, have been identified in all domains of life except eukaryotic nuclei. Controversially, the Mus81-Eme1 endonuclease has been proposed to be an example of a eukaryotic nuclear resolvase. However, hitherto little or no HJ cleavage has been detected in recombinant preparations of Mus81-Eme1. Here, we report the purification of active forms of recombinant Schizosaccharomyces pombe Mus81-Eme1 and Saccharomyces cerevisiae Mus81-Mms4, which display robust HJ cleavage in vitro, which, in the case of Mus81-Eme1, is as good as the archetypal HJ resolvase RuvC in single turnover kinetic analysis. We also present genetic evidence that suggests that this activity might be utilised as a back-up to Mus81-Eme1's main activity of cleaving nicked HJs during meiosis in S. pombe.     

Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells

Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3'-flap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1-/- embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity.     

Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease

The DNA structure-selective endonuclease Mus81-Mms4/Eme1 is a context-specific recombination factor that supports DNA replication, but is not essential for DSB repair in Saccharomyces cerevisiae. We overexpressed Mus81-Mms4 in S. cerevisiae, purified the heterodimer to apparent homogeneity, and performed a classical enzymological characterization. Kinetic analysis (k(cat), K(M)) demonstrated that Mus81-Mms4 is catalytically active and identified three substrate classes in vitro. Class I substrates reflect low K(M) (3-7 nM) and high k(cat) ( approximately 1 min(-1)) and include the nicked Holliday junction, 3'-flapped and replication fork-like structures. Class II substrates share low K(M) (1-6 nM) but low k(cat) (< or =0.3 min(-1)) relative to Class I substrates and include the D-loop and partial Holliday junction. The splayed Y junction defines a class III substrate having high K(M) ( approximately 30 nM) and low k(cat) (0.26 min(-1)). Holliday junctions assembled from oligonucleotides with or without a branch migratable core were negligibly cut in vitro. We found that Mus81 and Mms4 are phosphorylated constitutively and in the presence of the genotoxin MMS. The endogenous complex purified in either modification state is negligibly active on Holliday junctions. Hence, Holliday junction incision activity in vitro cannot be attributed to the Mus81-Mms4 heterodimer in isolation.     

Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells

The Mus81–Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3′-flap structures and aberrant replication fork structures. Mus81^−/− mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G₂/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.     

The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday junction resolution and crossover formation during meiosis.     

Swi5 acts in meiotic DNA joint molecule formation in Schizosaccharomyces pombe

Previously isolated Schizosaccharomyces pombe swi5 mutants are defective in mitotic mating-type switching and in repair of meiotic recombination-related DNA double-strand breaks. Here, we identify the swi5 gene, which encodes an 85-amino-acid polypeptide, similar to Sae3 of Saccharomyces cerevisiae, with an N-terminal predicted coiled-coil domain. A swi5 complete deletion mutant had normal mitotic growth rate but was hypersensitive to DNA-damaging agents and defective in mating-type switching. In meiosis, recombinant frequencies were reduced by a factor of approximately 10. The swi5 deletion strongly reduced the viable spore yields of mutants lacking Rhp55 or Rhp57, proteins thought to aid joint molecule formation. Furthermore, the swi5 deletion strongly suppressed the low viable spore yield of mutants lacking Mus81*Eme1, which resolves joint molecules such as Holliday junctions. These and previous results indicate that the small Swi5 polypeptide acts in a branched pathway of joint molecule formation to repair meiotic DNA breaks.     

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81?CMms4/EME1, Slx1?CSlx4/BTBD12/MUS312, XPF?CERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.     

Interplay between Np95 and Eme1 in the DNA damage response

Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.