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.     

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 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.     

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.     

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli.

Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA. A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange. In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure. In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene. Here, we describe the DNA binding properties of RusA. We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA. However, it does bind weakly to linear duplex DNA. Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions. The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.     

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.     

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.     

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.     

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.     

Crystal structure of RuvC resolvase in complex with Holliday junction substrate

The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein–DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.     

Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.

The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino-acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.     

The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold

Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases. We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes. The structure suggests that dimer formation is essential for Mg(2+) cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding. Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.     

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.     

The RuvABC resolvasome.

The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure-specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co-operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure-specific binding by RuvA.     

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.     

Cleavage of holliday junctions by the Escherichia coli RuvABC complex

The Escherichia coli RuvABC proteins process recombination intermediates during genetic recombination and recombinational repair. Although early biochemical studies indicated distinct RuvAB-mediated branch migration and RuvC-mediated Holliday junction resolution reactions, more recent studies have shown that the three proteins act together as a "resolvasome" complex. In this work we have used recombination intermediates made by RecA to determine whether the RuvAB proteins affect the sequence specificity of the RuvC resolvase. We find that RuvAB proteins do not alter significantly the site specificity of RuvC-dependent cleavage, although under certain conditions, they do affect the efficiency of cleavage at particular sites. The presence of RecA also influences cleavage at some sites. We also show that the RuvAB proteins act upon transient strand exchange intermediates made using substrates that have the opposite polarity of those preferred by RecA. Together, our results allow us to develop further a model for the recombinational repair of DNA lesions that lead to the formation of post-replication gaps during DNA replication. The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5'-(A/T)TT downward arrow(G/C)-3'; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation.     

Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations.

The ruvA, ruvB, and ruvC genes of Escherichia coli provide activities that catalyze branch migration and resolution of Holliday junction intermediates in recombination. Mutation of any one of these genes interferes with recombination and reduces the ability of the cell to repair damage to DNA. A suppressor of ruv mutations was identified on the basis of its ability to restore resistance to mitomycin and UV light and to allow normal levels of recombination in a recBC sbcBC strain carrying a Tn10 insertion in ruvA. The mutation responsible was located at 12.5 min on the genetic map and defines a new locus which has been designated rus. The rus suppressor works just as well in recBC sbcA and rec+ sbc+ backgrounds and is not allele specific. Mutations in ruvB and ruvC are suppressed to an intermediate level, except when ruvA is also inactive, in which case suppression is complete. In all cases, suppression depends on RecG protein, a DNA-dependent ATPase that catalyzes branch migration of Holliday junctions. The rus mutation activates an additional factor that probably works with RecG to process Holliday junction intermediates independently of the RuvAB and RuvC proteins. The possibility that this additional factor is a junction-specific resolvase is discussed.     

Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes

Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81–Mms4/MUS81–EME1, Yen1/GEN1, and Slx1–Slx4/SLX1–SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.     

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.     

Activation of RuvC Holliday junction resolvase in vitro.

The Escherichia coli RuvC protein is an endonuclease that resolves Holliday junctions. In vitro, the protein shows efficient structure-specific binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a synthetic junction through which a crossover can branch migrate through 26 bp and find that > or = 90% of the junctions were cleaved at one site. This observation of sequence-specific cleavage suggests that inefficient resolution may be due to DNA binding events which occur away from the cleavage site and are therefore non-productive. Holliday junction resolution by RuvC protein can be stimulated by a number of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (< or = 10), and (iii) elevated temperature. These observations may indicate that other proteins are required for efficient RuvC-mediated resolution.