The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks

Repair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81(-/-) Rad54(-/-) ES cells were as hypersensitive to ICL agents as Mus81(-/-) cells. We propose that Mus81-Eme1- and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.     

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.     

Identification and characterization of the human mus81-eme1 endonuclease

The faithful and complete replication of DNA is necessary for the maintenance of genome stability. It is known, however, that replication forks stall at lesions in the DNA template and need to be processed so that replication restart can occur. In fission yeast, the Mus81-Eme1 endonuclease complex (Mus81-Mms4 in Saccharomyces cerevisiae) has been implicated in the processing of aberrant replication intermediates. In this report, we identify the human homolog of the Schizosaccharomyces pombe EME1 gene and have purified the human Mus81-Eme1 heterodimer. We show that Mus81-Eme1 is an endonuclease that exhibits a high specificity for synthetic replication fork structures and 3'-flaps in vitro. The nuclease cleaves Holliday junctions inefficiently ( approximately 75-fold less than flap or fork structures), although cleavage can be increased 6-fold by the presence of homologous sequences previously shown to permit base pair "breathing." We conclude that human Mus81-Eme1 is a flap/fork endonuclease that is likely to play a role in the processing of stalled replication fork intermediates.     

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.     

Synthetic lethality of Drosophila in the absence of the MUS81 endonuclease and the DmBlm helicase is associated with elevated apoptosis

Mus81-Mms4 (Mus81-Eme1 in some species) is a heterodimeric DNA structure-specific endonuclease that has been implicated in meiotic recombination and processing of damaged replication forks in fungi. We generated and characterized mutations in Drosophila melanogaster mus81 and mms4. Unlike the case in fungi, we did not find any role for MUS81-MMS4 in meiotic crossing over. A possible role for this endonuclease in repairing double-strand breaks that arise during DNA replication is suggested by the finding that mus81 and mms4 mutants are hypersensitive to camptothecin; however, these mutants are not hypersensitive to other agents that generate lesions that slow or block DNA replication. In fungi, mus81, mms4, and eme1 mutations are synthetically lethal with mutations in genes encoding RecQ helicase homologs. Similarly, we found that mutations in Drosophila mus81 and mms4 are synthetically lethal with null mutations in mus309, which encodes the ortholog of the Bloom Syndrome helicase. Synthetic lethality is associated with high levels of apoptosis in proliferating tissues. Lethality and elevated apoptosis were partially suppressed by a mutation in spn-A, which encodes the ortholog of the strand invasion protein Rad51. These findings provide insights into the causes of synthetic lethality.     

The novel gene mus7(+) is involved in the repair of replication-associated DNA damage in fission yeast

The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Deltamus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Deltamus7 and Deltamus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Deltamus7 and Deltamus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Deltamus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81.     

Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks

Cells of all living organisms have evolved complex mechanisms that serve to stabilise, repair and restart stalled, blocked and broken replication forks. The heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease appears to play an important role(s) in homologous recombination-mediated processing of such perturbed forks. This enzyme has been implicated in the cleavage of stalled and blocked replication forks to initiate recombination, as well as in the processing of recombination intermediates that result from repairing damaged forks. In this review we assess the biochemical and genetic evidence for the mitotic role of Mus81-Eme1/Mms4 at replication forks and in repairing post-replication DNA damage. Mus81 appears to act when replication is impeded by genotoxins or by impairment of the replication machinery, or when arrested replication forks are not adequately protected. We discuss how its action is regulated by the S-phase cell cycle checkpoint, depending on the nature of the stalled or damaged fork. We also present a new way in which Mus81 may limit crossing over during the repair of post-replication gaps, and explore Mus81's interplay with other components of the recombination machinery, including the RecQ helicases that also play important roles in processing replication and recombination intermediates.     

Cell cycle-dependent regulation of the nuclease activity of Mus81-Eme1/Mms4

The conserved heterodimeric endonuclease Mus81-Eme1/Mms4 plays an important role in the maintenance of genomic integrity in eukaryotic cells. Here, we show that budding yeast Mus81-Mms4 is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this protein occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the function of the Mus81-Mms4 complex. Consistently, a phosphorylation-defective mms4 mutant shows highly reduced nuclease activity and increases the sensitivity of cells lacking the RecQ-helicase Sgs1 to various agents that cause DNA damage or replicative stress. The mode of regulation of Mus81-Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81-Mms4 activity provides a safeguard mechanism to resolve DNA intermediates that may remain after replication and require processing before mitosis.     

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.     

Mms22 preserves genomic integrity during DNA replication in Schizosaccharomyces pombe

The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important but poorly understood role in preservation of genome integrity. Here we describe a novel gene in Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22 functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability. There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to maintain genomic integrity in a pathway involving Mus81-Eme1.     

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.     

Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease

Correct replication of the genome and protection of its integrity are essential for cell survival. In a high-throughput screen studying H2AX phosphorylation, we identified Wee1 as a regulator of genomic stability. Wee1 down-regulation not only induced H2AX phosphorylation but also triggered a general deoxyribonucleic acid (DNA) damage response (DDR) and caused a block in DNA replication, resulting in accumulation of cells in S phase. Wee1-deficient cells showed a decrease in replication fork speed, demonstrating the involvement of Wee1 in DNA replication. Inhibiting Wee1 in cells treated with short treatment of hydroxyurea enhanced the DDR, which suggests that Wee1 specifically protects the stability of stalled replication forks. Notably, the DDR induced by depletion of Wee1 critically depends on the Mus81-Eme1 endonuclease, and we found that codepletion of Mus81 and Wee1 abrogated the S phase delay. Importantly, Wee1 and Mus81 interact in vivo, suggesting direct regulation. Altogether, these results demonstrate a novel role of Wee1 in controlling Mus81 and DNA replication in human 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.     

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.     

Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM helicase

The Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2^CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1^BLM helicase. Here we identify Rad3^ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1^BLM. We show that direct phosphorylation of Eme1 by Rad3^ATR is essential for catalytic stimulation of Mus81-Eme1. Chk1-mediated phosphorylation also contributes to the stimulation of Mus81-Eme1 when combined with phosphorylation of Eme1 by Rad3^ATR. Both Rad3^ATR- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1^BLM and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1Δ cell viability. Our findings unravel an elaborate regulatory network that relies on the poorly structured N-terminal domain of Eme1 and which is essential for the vital functions Mus81-Eme1 fulfills in absence of Rqh1^BLM.     

The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism

Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.     

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-mediated DNA cleavage resolves replication forks stalled by topoisomerase I-DNA complexes

Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1-DNA cleavage complexes (Top1cc's) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc's. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.     

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks.Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.