T4 endonuclease VII cleaves holliday structures

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.     

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.     

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.     

Effect of DNA structure and nucleotide sequence on Holliday junction resolution by a Saccharomyces cerevisiae endonuclease

Previous studies have demonstrated that mitotic Saccharomyces cerevisiae cells contain an endonuclease that cleaves Holliday junctions. In this paper, the cleavage of a number of model branched substrates has been characterized in detail. Three-armed Y-branched molecules were not substrates for the enzyme. Holliday junction substrates constructed from wild-type lambda att sites were resolved in a concerted reaction by paired single-strand breaks that contained 5'-phosphate and 3'-hydroxyl groups and were often symmetrically related. Holliday junctions were also constructed using DNAs derived from lambda safG and safT mutants to alter the nucleotide sequence immediately flanking the cross-strand exchange. These one to six base-pair changes in nucleotide sequence were observed to have dramatic effects on both the directionality and rate of resolution. More than 90% of wild-type junctions were cleaved in only one direction, while Holliday junctions composed of safT DNA were cleaved equally in both possible directions. Hybrid junctions composed of half wild-type DNA and half safG DNA were cleaved in the same orientation as the wild-type junction but at one-seventh of the rate, while junctions constructed completely from safG DNA were not cleaved at all. The cleavage sites were mapped at the nucleotide level and the locations of the paired nicks made by the endonuclease were also found to be affected by the sequence of the substrates and in such a way as to account for the directionality of cleavage. These results have important consequences for the interpretation of genetic experiments, since they provide biochemical evidence that some of the non-random nature of genetic recombination might be due to non-randomly distributed resolution processes.     

Resolution of model Holliday junctions by yeast endonuclease: effect of DNA structure and sequence.

The resolution of Holliday junctions in DNA involves specific cleavage at or close to the site of the junction. A nuclease from Saccharomyces cerevisiae cleaves model Holliday junctions in vitro by the introduction of nicks in regions of duplex DNA adjacent to the crossover point. In previous studies [Parsons and West (1988) Cell, 52, 621-629] it was shown that cleavage occurred within homologous arm sequences with precise symmetry across the junction. In contrast, junctions with heterologous arm sequences were cleaved asymmetrically. In this work, we have studied the effect of sequence changes and base modification upon the site of cleavage. It is shown that the specificity of cleavage is unchanged providing that perfect homology is maintained between opposing arm sequences. However, in the absence of homology, cleavage depends upon sequence context and is affected by minor changes such as base modification. These data support the proposed mechanism for cleavage of a Holliday junction, which requires homologous alignment of arm sequences in an enzyme--DNA complex as a prerequisite for symmetrical cleavage by the yeast endonuclease.     

Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage

In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.     

Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions

We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions. A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides. Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands. In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment. High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction. The synthetic Holliday junction was found to be a specific inhibitor of a S. cerevisiae enzyme that catalyzes the cleavage of Holliday junctions. Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1. Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex. The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays.     

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.     

High affinity of endonuclease VII for the Holliday structure containing one nick ensures productive resolution

During homologous recombination, genetic information is physically exchanged between parental DNAs via crossing single strands of the same polarity within a four-way DNA junction called a Holliday structure. This process is terminated by the endonucleolytic activity of resolvases, which convert the four-way DNA back to two double strands. To achieve productive resolution, the two subunits of the dimeric enzymes introduce two single-strand cuts positioned symmetrically in opposite strands across the DNA junction. Covalently linked dimers of endonuclease VII from phage T4, whether a homodimer with two or a heterodimer with only one functional catalytic centre, reacted with a synthetic cruciform DNA to form a DNA-enzyme complex immediately after addition of the enzyme. Analysis of the complexes from both reactions revealed that the bound junction contained one nick. While the active homodimer processed this nicked junction consecutively to duplex DNAs by making the second cut, the complex with the heterodimer stayed stable for the whole reaction time. Thus the high affinity of endonuclease VII for the junction containing one nick is part of the mechanism to ensure productive resolution of Holliday structures, by giving the enzyme time to make the second cut, whereupon the complex dissociates into the two duplex DNAs and the free enzyme.     

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.     

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I

Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.     

The endonuclease Hje catalyses rapid, multiple turnover resolution of Holliday junctions

Holliday junction-resolving enzymes are ubiquitous, structure-specific endonucleases that resolve four-way DNA junctions by the introduction of paired nicks in opposing strands, and are required for homologous recombination, double-strand break repair, recombination-dependent restart of stalled or collapsed DNA replication forks, and phage DNA processing. Here, we present the first steady-state kinetic characterisation of a junction-resolving enzyme; the Hje endonuclease from Sulfolobus solfataricus. We demonstrate that substrate turnover by Hje is sequence-independent and limited largely by the rate of cleavage of the phosphodiester bonds of the bound Holliday junction substrate, rather than substrate association or product dissociation. Reaction rates under multiple turnover conditions compare favourably with type II restriction enzymes. These properties, coupled with a high level of specificity for four-way junctions over all other DNA substrates, make Hje a suitable enzyme for applications requiring the detection and cleavage of Holliday junctions in vitro.     

Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII.

In common with a number of other DNA junction-resolving enzymes, endonuclease VII of bacteriophage T4 binds to a four-way DNA junction as a dimer, and cleaves two strands of the junction. We have used a supercoil-stabilized cruciform substrate to probe the simultaneity of cleavage at the two sites. Active endonuclease VII converts the supercoiled circular DNA directly into linear product, indicating that the two cleavage reactions must occur within the lifetime of the protein-junction complex. By contrast, a heterodimer of active enzyme and an inactive mutant endonuclease VII leads to the formation of nicked circular product, showing that the subunits operate fully independently.     

Biochemical characterization of a structure-specific resolving enzyme from Sulfolobus islandicus rod-shaped virus 2

Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.     

Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.     

An archaeal Holliday junction resolving enzyme from Sulfolobus solfataricus exhibits unique properties

The rearrangement and repair of DNA by homologous recombination often involves the creation of Holliday junctions, which must be cleaved by junction-specific endonucleases to yield recombinant duplex DNA products. Holliday junction resolving enzymes are a ubiquitous class of proteins with diverse structural and mechanistic characteristics. We have characterised an endonuclease (Hje) from the thermophilic crenarchaeote Sulfolobus solfataricus that exhibits a high degree of specificity for Holliday junctions via an apparently novel mechanism. Hje resolves four-way DNA junctions by the introduction of paired nicks in a reaction that is independent of the local nucleotide sequence, but is restricted solely to strands that are continuous in the stacked-X form of the junction. Three-way DNA junctions are cleaved only when the presence of a bulge in one strand allows the junction to stack in an analogous manner to four-way junctions. These properties differentiate Hje from all other known junction resolving enzymes.     

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.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.     

Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro.

Heteroduplex DNAs with single-stranded loops of 51 nt or 8 nt were constructed in vitro and used in reactions with purified endonuclease VII (endo VII) from phage T4. The enzyme makes double-strand breaks by introducing pairs of staggered nicks flanking the loops. Regardless of loop-size the nicking sites map exclusively at the 3' side of the loop in the looping strand and at the 3' side of the base of the loop in the non-looping strand. The number of potential cleavage sites is small (less than 5) and their distribution depends on DNA sequence. The two closest staggered nicks are 4 bp apart, 2 bp on either side of the loop. Nicking always occurs in the double-stranded part of the molecules; the single-stranded loops are not attacked by endo VII. The nicks are introduced in a stepwise fashion and selection of the strand for the first nick depends on the sequence of 31 base pairs flanking the loops.     

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.