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.     

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.     

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.     

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.     

New insight into the recognition of branched DNA structure by junction-resolving enzymes

Junction-resolving enzymes are nucleases that exhibit structural selectivity for the four-way (Holliday) junction in DNA. In general, these enzymes both recognize and distort the structure of the junction. New insight into the molecular recognition processes has been provided by two recent co-crystal structures of resolving enzymes bound to four-way DNA junctions in highly contrasting ways. T4 endonuclease VII binds the junction in an open conformation to an approximately flat binding surface whereas T7 endonuclease I envelops the junction, which retains a much more three-dimensional structure. Both proteins make contacts with the DNA backbone over an extensive area in order to generate structural specificity. The comparison highlights the versatility of Holliday junction resolution, and extracts some general principles of recognition.     

DNA branch nuclease activity of vaccinia A22 resolvase

DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.     

Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively‐charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild‐type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.     

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.     

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

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

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.     

Interactions between lambda Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution

lambda Site-specific recombination proceeds via two sequential single-strand exchanges that first generate and then resolve a Holliday recombination intermediate. The resolution of artificial Holliday junctions (chi-forms) is well suited to studying the mechanisms involved in reciprocal strand exchange because the linear products of this reaction are stable and easily quantitated. To study the interactions between Int molecules bound at the sites of strand exchange, artificial Holliday junctions containing only the seven base-pair overlap region and the four core-type Int binding sites were used as a model system. In vitro resolution of these structures yields products of both top- and bottom-strand exchange. An abortive product resulting from simultaneous cleavage of the top and bottom strands also occurs at low frequency. Inactivation of one of the four Int binding sites by multiple base substitutions does not significantly affect the efficiency of resolution but has a dramatic effect on the directionality, i.e. the choice of top- or bottom-strand exchange. When any two of the four core-type sites are similarly inactivated, strand exchange is very inefficient and the amount of aberrant cleavage is somewhat greater than for the Holliday junction with four intact Int binding sites. Analysis of the resolution products of Holliday junctions with various combinations of defective Int binding sites leads to the following conclusions: (1) three functional core-type Int binding sites are necessary and sufficient for a strand exchange; (2) the Int molecules that are partners in a strand exchange interact with Int bound to a "cross-core" site that is not directly involved in carrying out the reaction; (3) Int molecules bound to the core-type sites interact in a way that reduces the occurrence of abortive double-strand cleavage events.     

Cruciform-resolvase interactions in supercoiled DNA

T4 endonuclease VII, which cleaves Holliday-like junctions in DNA, specifically cleaves short inverted repeats in supercoiled plasmids. These sequences are subject to site-specific cleavage by single-strand-specific nucleases, and cruciform formation has been suggested as an explanation for this observation. This proposal is greatly strengthened by the present data, since a formal analogy between cruciform structures and Holliday junctions exists. Resolution of a variety of unrelated cruciform sequences demonstrates that the cleavage process results in a linear molecule with hairpin ends and single ligatable nicks at positions corresponding to the stem-base of the cruciform. In two examples mapped in detail, the cleavages are exclusively introduced at two or three nucleotides from the end of the symmetric sequence at the 5' side on each strand. These studies demonstrate the potential of endonuclease VII as a probe of cruciform structure and the utility of short cruciform structures as Holliday junction models.     

Human Mus81-associated endonuclease cleaves Holliday junctions in vitro.

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

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 phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.     

Dissociation of synthetic Holliday junctions by E. coli RecG protein.

The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities than can promote branch migration of Holliday junctions in recombination and DNA repair.     

Molecular mechanisms of holliday junction processing in Escherichia coli

Recent genetic and biochemical studies revealed the mechanisms of late stage of homologous recombination in E. coli. A central intermediate of recombination called “Holliday structure”, in which two homologous duplex DNA molecules are linked by a single-stranded crossover, is formed by the functions of RecA and several other proteins. The products of the ruvA and ruvB genes, which constitute an SOS regulated operon, form a functional complex that promotes migration of Holliday junctions by catalyzing strand exchange reaction, thus enlarging the heteroduplex region. RuvA is a DNA-binding protein specific for these junctions, and RuvB is a motor molecule for branch migration providing energy by hydrolyzing ATP. The product of the ruvC gene, which is not regulated by the SOS system, resolves Holliday junctions by introducing nicks at or near the crossover junction in strands with the same polarity at the same sites. The recombination reaction is completed by sealing the nicks with DNA ligase, resulting in spliced or patched recombinants. The product of the recG gene provides an alternative route for resolving Holliday junctions. RecG has been proposed to promote branch migration in the opposite direction to that promoted by RecA protein. The atomic structure of RuvC protein revealed by crystallographic study, when combined with mutational analysis of RuvC, provides mechanistic insights into the interactions of RuvC with Holliday junction.     

Nucleic acid crystallography: current progress

Fifty years after the publication of the DNA double helix model by Watson and Crick, new nucleic acid structures keep emerging at an ever-increasing rate. The past three years have brought a flurry of new oligonucleotide structures, including those of a Hoogsteen-paired DNA duplex, Holliday junctions, DNA-drug complexes, quadruplexes, a host of RNA motifs and various nucleic acid analogues. Major advances were also made in terms of the structure and function of catalytic RNAs. These range from improved models of the phosphodiester cleavage reactions catalyzed by the hairpin and hepatitis delta virus ribozymes to the visualization of a complete active site of a group I self-splicing intron with bound 5'- and 3'-exons. These triumphs are complemented by a refined understanding of cation-nucleic-acid interactions and new routes to the generation of derivatives for phasing of DNA and RNA structures.