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.     

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.     

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.     

Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.     

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.     

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.     

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.     

Tipin Functions in the Protection against Topoisomerase I Inhibitor

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.     

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.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.     

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.     

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.     

Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation

The DNA-damage checkpoint kinase Chk1 is essential in higher eukaryotes due to its role in maintaining genome stability in proliferating cells. CHK1 gene deletion is embryonically lethal, and Chk1 inhibition in replicating cells causes cell-cycle defects that eventually lead to perturbed replication and replication-fork collapse, thus generating endogenous DNA damage. What is the cause of replication-fork collapse when Chk1 is inactivated, however, remains poorly understood. Here, we show that generation of DNA double-strand breaks at replication forks when Chk1 activity is compromised relies on the DNA endonuclease complex Mus81/Eme1. Importantly, we show that Mus81/Eme1-dependent DNA damage--rather than a global increase in replication-fork stalling--is the cause of incomplete replication in Chk1-deficient cells. Consequently, Mus81/Eme1 depletion alleviates the S-phase progression defects associated with Chk1 deficiency, thereby increasing cell survival. Chk1-mediated protection of replication forks from Mus81/Eme1 even under otherwise unchallenged conditions is therefore vital to prevent uncontrolled fork collapse and ensure proper S-phase progression in human cells.     

RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1? rnh201? cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1? rnh201? cells, but RNase H is not generally required for efficient DSB repair.     

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.     

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.     

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.