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.     

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.     

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.     

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.     

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.     

Crystal structures of the structure‐selective nuclease Mus81‐Eme1 bound to flap DNA substrates

The Mus81‐Eme1 complex is a structure‐selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross‐links, replication fork collapse, or double‐strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81‐Eme1, we determined crystal structures of human Mus81‐Eme1 bound to various flap DNA substrates. Mus81‐Eme1 undergoes gross substrate‐induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre‐ and post‐nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post‐nick DNA. These features are crucial for comprehensive protein‐DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81‐Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81‐Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends.     

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.     

Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity but does not promote tumorigenesis

The Mus81-Eme1 endonuclease is implicated in the efficient rescue of broken replication forks in Saccharomyces cerevisiae and Schizosaccharomyces pombe. We have used gene targeting to study the function of the Mus81-Eme1 endonuclease in mammalian cells. Mus81-deficient mice develop normally and are fertile. Surprisingly, embryonic fibroblasts from Mus81(-/-) animals fail to proliferate in vitro. This proliferation defect can be rescued by expression of the papillomavirus E6 protein that promotes degradation of p53. When grown in culture, Mus81(-/-) cells have elevated levels of DNA damage, acquire chromosomal aberrations, and are hypersensitive to agents that generate DNA cross-links. In contrast to the situation in yeast, murine Mus81 is not required for replication restart following camptothecin treatment. Mus81(-/-) mice and cells are hypersensitive to DNA cross-linking agents. Cross-link-induced double-strand break formation is normal in Mus81(-/-) cells, but the resolution of repair intermediates is not. The persistence of Rad51 foci in Mus81(-/-) cells suggests that Mus81 acts at a late step in the repair of cross-link-induced lesions. Despite these defects, Mus81(-/-) mice do not show increased predisposition to lymphoma or any other malignancy in the first year of life.     

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.     

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.     

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.     

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.     

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.     

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.     

Human MUS81 complexes stimulate flap endonuclease 1

The yeast heterodimeric Mus81-Mms4 complex possesses a structure-specific endonuclease activity that is critical for the restart of stalled replication forks and removal of toxic recombination intermediates. Previously, we reported that Mus81-Mms4 and Rad27 (yeast FEN1, another structure-specific endonuclease) showed mutual stimulation of nuclease activity. In this study, we investigated the interactions between human FEN1 and MUS81-EME1 or MUS81-EME2, the human homologs of the yeast Mus81-Mms4 complex. We found that both MUS81-EME1 and MUS81-EME2 increased the activity of FEN1, but FEN1 did not stimulate the activity of MUS81-EME1/EME2. The MUS81 subunit alone and its N-terminal half were able to bind to FEN1 and stimulate its endonuclease activity. A truncated FEN1 fragment lacking the C-terminal region that retained catalytic activity was not stimulated by MUS81. Michaelis-Menten kinetic analysis revealed that MUS81 increased the interaction between FEN1 and its substrates, resulting in increased turnover. We also showed that, after DNA damage in human cells, FEN1 co-localizes with MUS81. These findings indicate that the human proteins and yeast homologs act similarly, except that the human FEN1 does not stimulate the nuclease activities of MUS81-EME1 or MUS81-EME2. Thus, the mammalian MUS81 complexes and FEN1 collaborate to remove the various flap structures that arise during many DNA transactions, including Okazaki fragment processing.     

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.     

Srs2 promotes Mus81–Mms4-mediated resolution of recombination intermediates

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81–Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81–Mms4. In this study, we show that the Srs2 and Mus81–Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81–Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81–Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81–Mms4 to cleave DNA. Concomitantly, Mus81–Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81–Mms4 and Srs2 in processing of recombination as well as replication intermediates.     

Genetic and functional interactions between Mus81–Mms4 and Rad27

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.     

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.     

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.