The stacked-X DNA Holliday junction and protein recognition

The crystal structure of the four-stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open-X form of the junction seen in all protein complexes, but the more compact stacked-X structure observed in free DNA. The structures of the stacked-X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked-X junctions, with the junction cross-over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked-X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition.     

Structural dynamics of individual Holliday junctions

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.     

A method for preparing genomic DNA that restrains branch migration of Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. This four-stranded DNA structure is capable of spontaneous branch migration, and is lost during standard DNA extraction protocols. In order to isolate and characterize recombination intermediates that contain Holliday junctions, we have developed a rapid protocol that restrains branch migration of four-way DNA junctions. The cationic detergent hex-adecyltrimethylammonium bromide is used to lyse cells and precipitate DNA. Manipulations are performed in the presence of the cations hexamine cobalt(III) or magnesium, which stabilize Holliday junctions in a stacked-X configuration that branch migrates very slowly. This protocol was evaluated using a sensitive assay for spontaneous branch migration, and was shown to preserve both artificial Holliday junctions and meiotic recombination intermediates containing four-way junctions.     

The crystal structures of DNA Holliday junctions.

Nearly 40 years ago, Holliday proposed a four-stranded complex or junction as the central intermediate in the general mechanism of genetic recombination. During the past two years, six single-crystal structures of such DNA junctions have been determined by three different research groups. These structures all essentially adopt the antiparallel stacked-X conformation, but can be classified into three distinct categories: RNA-DNA junctions; ACC trinucleotide junctions; and drug-induced junctions. Together, these structures provide insight into how local and distant interactions help to define the detailed and general physical features of Holliday junctions at the atomic level.     

Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy

The four-way DNA (Holliday) junction is an essential intermediate in DNA recombination, and its dynamic characteristics are likely to be important in its cellular processing. In our previous study we observed transitions between two antiparallel stacked conformations using a single-molecule fluorescence approach. The magnesium concentration-dependent rates of transitions between stacking conformers suggested that an unstacked open structure, which is stable in the absence of metal ions, is an intermediate. Here, we sought to detect possible rare species such as open and parallel conformations and further characterized ionic effects. The hypothesized open intermediate cannot be resolved directly due to the limited time resolution and sensitivity, but our study suggests that the open form is achieved very frequently, hundreds of times per second under physiologically relevant conditions. Therefore despite being a minority species, its frequent formation raises the probability that it could become stabilized by protein binding. By contrast, we cannot detect even a transient existence of the junctions in a parallel form, and the probability of such forms with a lifetime greater than 5 ms is less than 0.01%. Stacking conformer transitions are observable in the presence of sodium or hexammine cobalt (III) ions as well as magnesium ions, but the transition rates are higher for lower valence ions at the same concentrations. This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms.     

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.     

Conformational and hydration effects of site-selective sodium,calcium and strontium ion binding to the DNA Holliday junction structure d(TCGGTACCGA)(4)

The role of metal ions in determining the solution conformation of the Holliday junction is well established, but to date the picture of metal ion binding from structural studies of the four-way DNA junction is very incomplete. Here we present two refined structures of the Holliday junction formed by the sequence d(TCGGTACCGA) in the presence of Na(+) and Ca(2+), and separately with Sr(2+) to resolutions of 1.85A and 1.65A, respectively. This sequence includes the ACC core found to promote spontaneous junction formation, but its structure has not previously been reported. Almost complete hydration spheres can be defined for each metal cation. The Na(+) sites, the most convincing observation of such sites in junctions to date, are one on either face of the junction crossover region, and stabilise the ordered hydration inside the junction arms. The four Ca(2+) sites in the same structure are at the CG/CG steps in the minor groove. The Sr(2+) ions occupy the TC/AG, GG/CC, and TA/TA sites in the minor groove, giving ten positions forming two spines of ions, spiralling through the minor grooves within each arm of the stacked-X structure. The two structures were solved in the two different C2 lattices previously observed, with the Sr(2+) derivative crystallising in the more highly symmetrical form with two-fold symmetry at its centre. Both structures show an opening of the minor groove face of the junction of 8.4 degrees in the Ca(2+) and Na(+) containing structure, and 13.4 degrees in the Sr(2+) containing structure. The crossover angles at the junction are 39.3 degrees and 43.3 degrees, respectively. In addition to this, a relative shift in the base pair stack alignment of the arms of 2.3A is observed for the Sr(2+) containing structure only. Overall these results provide an insight into the so-far elusive stabilising ion structure for the DNA Holliday junction.     

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.     

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.     

Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme

The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.     

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.     

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.     

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.     

Molecular dynamics of a DNA Holliday junction: the inverted repeat sequence d(CCGGTACCGG)₄

All-atom molecular dynamics (MD) computer simulations have been applied successfully to duplex DNA structures in solution for some years and found to give close accord with observed results. However, the MD force fields have generally not been parameterized against unusual DNA structures, and their use to obtain dynamical models for this class of systems needs to be independently validated. The four-way junction (4WJ), or Holliday junction, is a dynamic DNA structure involved in central cellular processes of homologous replication and double strand break repair. Two conformations are observed in solution: a planar open-X form (OPN) with a mobile center and four duplex arms, and an immobile stacked-X (STX) form with two continuous strands and two crossover strands, stabilized by high salt conditions. To characterize the accuracy of MD modeling on 4WJ, we report a set of explicit solvent MD simulations of ～100 ns on the repeat sequence d(CCGGTACCGG)₄ starting from the STX structure (PDB code 1dcw), and an OPN structure built for the same sequence. All 4WJ MD simulations converged to a stable STX structure in close accord with the crystal structure. Our MD beginning in the OPN form converts to the STX form spontaneously at both high and low salt conditions, providing a model for the conformational transition. Thus, these simulations provide a successful account of the dynamical structure of the STX form of d(CCGGTACCGG)₄ in solution, and provide new, to our knowledge, information on the conformational stability of the junction and distribution of counterions in the junction interior.     

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 Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.     

Visualization of novel simian virus 40 DNA recombination intermediates induced by ultraviolet light irradiation.

Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells. Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA. These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways. In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed. In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA. The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms. The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths. The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.     

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.     

Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.     

The efficiency of mispaired ligations by lambda integrase is extremely sensitive to context

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.