Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.     

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.     

Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four-way junction (X-DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X-DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.     

The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts.

The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.     

Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the RusA holliday junction resolvase

Holliday junctions are key intermediates in both homologous recombination and DNA repair, and are also formed from replication forks stalled at lesions in the template strands. Their resolution is critical for chromosome segregation and cell viability, and is mediated by a class of small, homodimeric endonucleases that bind the structure and cleave the DNA. All the enzymes studied require divalent metal ions for strand cleavage and their active centres are characterised by conserved aspartate/glutamate residues that provide ligands for metal binding. Sequence alignments reveal that they also contain a number of conserved basic residues. We used site-directed mutagenesis to investigate such residues in the RusA resolvase. RusA is a 120 amino acid residue polypeptide that can be activated in Escherichia coli to promote recombination and repair in the absence of the Ruv proteins. The RuvA, RuvB and RuvC proteins form a complex on Holliday junction DNA that drives coupled branch migration (RuvAB) and resolution (RuvC) reactions. In contrast to RuvC, the RusA resolvase does not interact directly with a branch migration motor, which simplifies analysis of its resolution activity. Catalysis depends on three highly conserved acidic residues (Asp70, Asp72 and Asp91) that define the catalytic centre. We show that Lys76, which is invariant in RusA sequences, is essential for catalysis, but not for DNA binding, and that an invariant asparagine residue (Asn73) is required for optimal activity. Analysis of DNA binding revealed that RusA may interact with one face of an open junction before manipulating its conformation in the presence of Mg(2+) as part of the catalytic process. A well-conserved arginine residue (Arg69) is linked with this critical stage. These findings provide the first insights into the roles played by basic residues in DNA binding and catalysis by a Holliday junction resolvase.     

Dissecting the resolution reaction of lambda integrase using suicide Holliday junction substrates.

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.     

The X philes: structure-specific endonucleases that resolve Holliday junctions

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.     

Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.     

Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex

CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae. Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively. Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner. This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange. From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage. These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes.     

Sequence and functional-group specificity for cleavage of DNA junctions by RuvC of Escherichia coli.

RuvC is the DNA junction-resolving enzyme of Escherichia coli. While the enzyme binds to DNA junctions independently of base sequence, it exhibits considerable sequence selectivity for the phosphodiester cleavage reaction. We have analyzed the sequence specificity using a panel of DNA junctions, measuring the rate of cleavage of each under single-turnover conditions. We have found that the optimal sequence for cleavage can be described by (A approximately T)TT downward arrow(C>G approximately A), where downward arrow denotes the position of backbone scission. Cleavage is fastest when the cleaved phosphodiester linkage is located at the point of strand exchange. However, cleavage is possible one nucleotide 3' of this position when directed by the sequence, with a rate that is 1 order of magnitude slower than the optimal. The maximum sequence discrimination occurs at the central TT in the tetranucleotide site, where any alteration of sequence results in a rate reduction of at least 100-fold and cleavage is undetectable for some changes. However, certain sequences in the outer nucleotides are strongly inhibitory to cleavage. Introduction of base analogues around the cleavage site reveals a number of important functional groups and suggests that major-groove contacts in the center of the tetranucleotide are important for the cleavage process. Since RuvC binds to all the variant junctions with very similar affinity, any contacts affecting the rate of cleavage must be primarily important in the transition state. Introduction of the optimal cleavage sequence into a three-way DNA junction led to relatively efficient cleavage by RuvC, at a rate only 3-fold slower than the optimal four-way junction. This is consistent with a protein-induced alteration in the conformation of the DNA.     

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.     

Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC

The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.     

Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2.

Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.     

Biological roles of the Escherichia coli RuvA, RuvB and RuvC proteins revealed

In Escherichia coli, the ruvA, ruvB and ruvC gene products are required for genetic recombination and the recombinational repair of DNA damage. New studies suggest that these three proteins function late in recombination and process Holliday junctions made by RecA protein-mediated strand exchange. In vitro, RuvA protein binds a Holliday junction with high affinity and, together with RuvB (an ATPase), promotes ATP-dependent branch migration of the junction leading to the formation of heteroduplex DNA. The third protein, RuvC, which acts independently of RuvA and RuvB, resolves recombination intermediates by specific endonucleolytic cleavage of the Holliday junction.     

Unraveling the late stages of recombinational repair: Metabolism of DNA junctions in Escherichia coli

DNA junctions are by-products of recombinational repair, during which a damaged DNA sequence, assisted by RecA filament, invades an intact homologous DNA to form a joint molecule. The junctions are three-strand or four-strand depending on how many single DNA strands participate in joint molecules. In E. coli, at least two independent pathways to remove the junctions are proposed to operate. One is via RuvAB-promoted migration of four-strand junctions with their subsequent resolution by RuvC. In vivo, RuvAB and RuvC enzymes might work in a single complex, a resolvasome, to clean DNA from used RecA filaments and to resolve four-strand junctions. An alternative pathway for junction removal could be via RecG-promoted branch migration of three-strand junctions, provided that an as yet uncharacterized endonuclease activity incises one of the strands in the joint molecules.     

GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.     

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.     

Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli.

The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products. RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange. The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions. RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration. Both RuvA and RecG form defined complexes with each of the junctions. The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG. Dissociation of the junction in the presence of ATP requires high levels of RuvAB. RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo. Both proteins also dissociate a Y-junction, which is consistent with helicase activity. However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.     

Parallel symmetric immobile DNA junctions as substrates for E. coli RuvC Holliday junction resolvase

RuvC is a well-characterized Holliday junction resolvase from E. coli. The presence of symmetry in its preferred recognition sequence leads to ambiguity in the position of the crossover point in the junction, because a symmetric junction can undergo branch migration. Symmetric immobile junctions are junctions that contain such symmetric sites, but are prevented from migrating by their physical characteristics. RuvC activity had been analyzed previously by traditional symmetric immobile junctions, in which the helix axes are held antiparallel in a double-crossover motif. Bowtie junctions are branched four-arm molecules containing 5',5' and 3',3' linkages at their crossover points. A new type of symmetric immobile junction can be made by flanking the crossover point of a Bowtie junction with a symmetric sequence. The junction is immobile because mobility would lead to pairing between parallel, rather than antiparallel, nucleotide pairs. In contrast to conventional Holliday junctions and their analogues, the Bowtie junction assumes a parallel, rather than antiparallel, helical domain conformation, offering a new type of substrate for Holliday junction resolvases. Here, we report the digestion of Bowtie junctions by RuvC. We demonstrate that Bowtie junctions can function as symmetric immobile junctions in this system. We also show that RuvC cleaves antiparallel junctions much more efficiently than parallel junctions, where the protein can bind (and cleave) only one site at a time. These data suggest that the presence of two binding sites leads to communication between the two subunits of the enzyme to increase its activity.     

p53 blocks RuvAB promoted branch migration and modulates resolution of Holliday junctions by RuvC

The Holliday junction is the central intermediate in homologous recombination. Branch migration of this four-stranded DNA structure is a key step in genetic recombination that affects the extent of genetic information exchanged between two parental DNA molecules. Here, we have constructed synthetic Holliday junctions to test the effects of p53 on both spontaneous and RuvAB promoted branch migration as well as the effect on resolution of the junction by RuvC. We demonstrate that p53 blocks branch migration, and that cleavage of the Holliday junction by RuvC is modulated by p53. These findings suggest that p53 can block branch migration promoted by proteins such as RuvAB and modulate the cleavage by Holliday junction resolution proteins such as RuvC. These results suggest that p53 could have similar effects on eukaryotic homologues of RuvABC and thus have a direct role in recombinational DNA repair.