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.     

The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1

Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression. Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs. Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1. Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system. These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding. However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes. We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.     

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.     

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.     

Promiscuous and lineage-specific roles of cell cycle regulators in haematopoiesis

DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.     

Repair and antirepair DNA helicases in Helicobacter pylori

Orthologs of RecG and RuvABC are highly conserved among prokaryotes; in Escherichia coli, they participate in independent pathways that branch migrate Holliday junctions during recombinational DNA repair. RecG also has been shown to directly convert stalled replication forks into Holliday junctions. The bacterium Helicobacter pylori, with remarkably high levels of recombination, possesses RecG and RuvABC homologs, but in contrast to E. coli, H. pylori RecG limits recombinational repair. We now show that the RuvABC pathway plays the prominent, if not exclusive, repair role. By introducing an E. coli resolvase (RusA) into H. pylori, the repair and recombination phenotypes of the ruvB mutant but not the recG mutant were improved. Our results indicate that RecG and RuvB compete for Holliday junction structures in recombinational repair, but since a classic RecG resolvase is absent from H. pylori, deployment of the RecG pathway is lethal. We propose that evolutionary loss of the H. pylori RecG resolvase provides an "antirepair" pathway allowing for selection of varied strains. Such competition between repair and antirepair provides a novel mechanism to maximize fitness at a bacterial population level.     

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.     

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.     

Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda

Rap endonuclease targets recombinant joint molecules arising from phage lambda Red-mediated genetic exchange. Previous studies revealed that Rap nicks DNA at the branch point of synthetic Holliday junctions and other DNA structures with a branched component. However, on X junctions incorporating a three base-pair core of homology or with a fixed crossover, Rap failed to make the bilateral strand cleavages characteristic of a Holliday junction resolvase. Here, we demonstrate that Rap can mediate symmetrical resolution of 50 bp and chi Holliday structures containing larger homologous cores. On two different mobile 50 bp junctions Rap displays a weak preference for cleaving the phosphodiester backbone between 5'-GC dinucleotides. The products of resolution on both large and small DNA substrates can be sealed by T4 DNA ligase, confirming the formation of nicked duplexes. Rap protein was also assessed for its capacity to influence the global conformation of junctions in the presence or absence of magnesium ions. Unlike the known Holliday junction binding proteins, Rap does not affect the angle of duplex arms, implying an unorthodox mode of junction binding. The results demonstrate that Rap can function as a Holliday junction resolvase in addition to eliminating other branched structures that may arise during phage recombination.     

Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate

Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair. This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme. F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a DeltaruvC strain and substantially resolves synthetic Holliday junctions in vitro. In the presence of 100 mm NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay. KMnO(4) was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC. Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.     

The RuvABC resolvase is indispensable for recombinational repair in sbcB15 mutants of Escherichia coli

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the (Delta)ruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC (Delta)ruvABC and sbcB (Delta)ruvABC mutants displayed higher recombination proficiency than the recBC sbcBC (Delta)ruvABC strain. The frequency of conjugational recombination observed with the sbcB (Delta)ruvABC strain was quite similar to that of the (Delta)ruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.     

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.     

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.     

Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells

During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.     

Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.     

Mus81-Eme1 are essential components of a Holliday junction resolvase.

Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.     

Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli.

Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.     

The ruv proteins of Thermotoga maritima: branch migration and resolution of Holliday junctions

In homologous recombination in bacteria, the RuvAB Holliday junction-specific helicase catalyzes Holliday junction branch migration, and the RuvC Holliday junction resolvase catalyzes formation of spliced or patched structures. RuvAB and RuvC from the hyperthermophile Thermotoga maritima were expressed in Escherichia coli and purified to homogeneity. An inverted repeat sequence with unique termini was produced by PCR, restriction endonuclease cleavage, and head-to-tail ligation. A second inverted repeat sequence was derived by amplification of a second template containing a three-nucleotide insertion. Reassociation products from a mixture of these two sequences were homoduplex linear molecules and heteroduplex heat-stable Holliday junctions, which acted as substrates for both T. maritima RuvAB and RuvC. The T. maritima RuvAB helicase catalyzed energy-dependent Holliday junction branch migration at 70 degrees C, leading to heteroduplex linear duplex molecules with two three-nucleotide loops. Either ATP or ATP gamma S hydrolysis served as the energy source. T. maritima RuvC resolved Holliday junctions at 70 degrees C. Remarkably, the cleavage site was identical to the preferred cleavage site for E. coli RuvC [(A/T)TT(downward arrow)(G/C)]. The conservation of function and the ease of purification of wild-type and mutant thermophilic proteins argues for the use of T. maritima proteins for additional biochemical and structural studies.     

Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions

Resolution of Holliday junction recombination intermediates in most Gram-negative bacteria is accomplished by the RuvC endonuclease acting in concert with the RuvAB branch migration machinery. Gram-positive species, however, lack RuvC, with the exception of distantly related orthologues from bacteriophages infecting Lactococci and Streptococci. We have purified one of these proteins, 67RuvC, from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between 67RuvC and Escherichia coli RuvC were noted, although both enzymes prefer to cleave 3' of thymidine residues. However, unlike its cellular counterpart, 67RuvC readily binds and cleaves a variety of branched DNA substrates in addition to Holliday junctions. Plasmids expressing 67RuvC induce chromosomal breaks, probably as a consequence of replication fork cleavage, and cannot be recovered from recombination-defective E. coli strains. Despite these deleterious effects, 67RuvC constructs suppress the UV light sensitivity of ruvA, ruvAB and ruvABC mutant strains confirming that the phage protein mediates Holliday junction resolution in vivo. The characterization of 67RuvC offers a unique insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination.     

Identification of RecQL1 as a Holliday junction processing enzyme in human cell lines

Homologous recombination provides an effective way to repair DNA double-strand breaks (DSBs) and is required for genetic recombination. During the process of homologous recombination, a heteroduplex DNA structure, or a ‘Holliday junction’ (HJ), is formed. The movement, or branch migration, of this junction is necessary for recombination to proceed correctly. In prokaryotes, the RecQ protein or the RuvA/RuvB protein complex can promote ATP-dependent branch migration of Holliday junctions. Much less is known about the processing of Holliday junctions in eukaryotes. Here, we identify RecQL1 as a predominant ATP-dependent, HJ branch migrator present in human nuclear extracts. A reduction in the level of RecQL1 induced by RNA interference in HeLa cells leads to an increase in sister chromatid exchange. We propose that RecQL1 is involved in the processing of Holliday junctions in human cells.