Direct evidence for spontaneous branch migration in antiparallel DNA Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms flanking a branch point. In naturally occurring Holliday junctions, the sequence flanking the branch point contains 2-fold (homologous) symmetry. As a consequence of this symmetry, the junction can undergo a conformational isomerization known as branch migration, which relocates the site of branching. In the absence of proteins and in the presence of Mg(2+), the four arms are known to stack in pairs, forming two helical domains whose orientations are antiparallel. Nevertheless, the mechanistic models proposed for branch migration are all predicated on a parallel alignment of helical domains. Here, we have used antiparallel DNA double crossover molecules to demonstrate that branch migration can occur in antiparallel Holliday junctions. We have constructed a DNA double crossover molecule with three crossover points. Two adjacent branch points in this molecule are flanked by symmetric sequences. The symmetric crossover points are held immobile by the third crossover point, which is flanked by asymmetric sequences. Restriction of the helices that connect the immobile junction to the symmetric junctions releases this constraint. The restricted molecule undergoes branch migration, even though it is constrained to an antiparallel conformation.     

The unusual and dynamic character of PX-DNA

PX-DNA is a four-stranded DNA structure that has been implicated in the recognition of homology, either continuously, or in an every-other-half-turn fashion. Some of the structural features of the molecule have been noted previously, but the structure requires further characterization. Here, we report atomic force microscopic characterization of PX molecules that contain periodically placed biotin groups, enabling the molecule to be labeled by streptavidin molecules at these sites. In comparison with conventional double stranded DNA and with antiparallel DNA double crossover molecules, it is clear that PX-DNA is a more dynamic structure. Furthermore, the spacing between the nucleotide pairs along the helix axis is shorter, suggesting a mixed B/A structure. Circular dichroism spectroscopy indicates unusual features in the PX molecule that are absent in both the molecules to which it is compared.     

No braiding of Holliday junctions in positively supercoiled DNA molecules

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.     

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.     

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.     

Construction and analysis of parallel and antiparallel Holliday junctions

The Holliday junction is a four-stranded DNA intermediate that arises during recombination reactions. We have designed and constructed a set of Holliday junction analogs that model each of the ideal conformations available to a 2-fold symmetric four-arm junction. The strategy used is to connect two arms of a junction molecule with a short tether of thymidines. These DNA molecules share a common core sequence but have different arms that are connected so that each molecule is constrained in either an antiparallel or a parallel structure. For tethered antiparallel molecules the identity of the crossover strands is determined by which arms are connected. Different arm connections gave molecules representing each of the two antiparallel crossover isomers. Two parallel molecules that differ in the length and position of the tether exhibit opposite biases in their choice of crossover strands. Thus, a physical constraint applied at a distance from the branch point can determine the conformation of a junction.     

The construction of a trefoil knot from a DNA branched junction motif

A DNA trefoil (3₁) knot has been constructed from a 104-nucleotide molecule whose strands form a 3-arm branched junction motif. This construction tests the notion that a node in a DNA knot can be equated with a half-turn of double-helical DNA, and is consistent with that concept. Of five 104-mer sequences tested, only one produces high yields of the target knot. The other molecules produce larger quantities of circular material and of a knot containing more nodes. The key features that differentiate the successful design from the others are (1) the ligation takes place in the linker region between helical domains and (2) only six nucleotide pairs are used for each of the double-helical arms of the junction. The successful design separates the double-helical regions from each other by a spacer containing two deoxythymidine nucleotides at the site of the branched junction. © 1994 John Wiley & Sons, Inc.     

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.     

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.     

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.     

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2)

The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG^5BrUACG)₂ and d(CGTACG)₂ have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).     

The flexibility of DNA double crossover molecules

Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. There are several types of double crossover molecules, differentiated by the relative orientations of their helix axes, parallel or antiparallel, and by the number of double helical half-turns (even or odd) between the two crossovers. They are found as intermediates in meiosis and they have been used extensively in structural DNA nanotechnology for the construction of one-dimensional and two-dimensional arrays and in a DNA nanomechanical device. Whereas the parallel double helical molecules are usually not well behaved, we have focused on the antiparallel molecules; antiparallel molecules with an even number of half-turns between crossovers (termed DAE molecules) produce a reporter strand when ligated, facilitating their characterization in a ligation cyclization assay. Hence, we have estimated the flexibility of antiparallel DNA double crossover molecules by means of ligation-closure experiments. We are able to show that these molecules are approximately twice as rigid as linear duplex DNA.     

Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg²⁺ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology.     

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.     

Different conformational families of pyrimidine.purine.pyrimidine triple helices depending on backbone composition.

Different helical conformations of DNA (D), RNA (R), and DNA.RNA (DR) hybrid double and triple helices have been detected using affinity cleavage analysis. Synthetic methods were developed to attach EDTA.Fe to a single nucleotide on RNA as well as DNA oligonucleotides. Cleavage patterns generated by a localized diffusible oxidant in the major groove on the pyrimidine strand of four purine.pyrimidine double helices consisting of all DNA, all RNA, and the corresponding hybrids reveal that the relative cleavage intensity shifts to the 5' end of the purine strand increasingly in the order: DD < DR < RD < RR. These results are consistent with models derived from structural studies. In six pyrimidine.purine.pyrimidine triple helices, the altered cleavage patterns of the Watson-Crick pyrimidine strands reveal at least two conformational families: (i) D + DD, R + DD, D + DR, and R + DR and (ii) R + RD and R + RR.     

Four-Stranded Intercalated Cytosine-Rich Molecules: Novel Insights into DNA Structure and Stability

Abstract

DNA fragments with stretches of cytosine residues can form four-stranded intercalated i-DNA molecules stabilized by hemiprotonated cytosine·cytosine⁺ (C·C⁺) base pairs. Intriguing features of this motif are the accomodation of base stacking that is unfavorable due to electrostatic repulsion and the close approach of phosphates in narrow grooves of the molecule. Unusual sources of stability in this structure involve sugar-base stacking and CH-O interribose short contacts between the backbones of adjacent strands.     

The stability of Seeman JX DNA topoisomers of paranemic crossover (PX) molecules as a function of crossover number

We use molecular dynamics simulations in explicit water and salt (Na⁺) to determine the effect of varying the number of crossover points on the structure and stability of the PX65 paranemic crossover DNA molecule and its JXM topoisomers (M denotes the number of missing crossover points), recently synthesized by the Seeman group at New York University. We find that PX65, with six crossover points, is the most stable, and that the stability decreases monotonically with the number of crossover points PX65 > JX1 > JX2 > JX3 > JX4, with 6, 5, 4, 3 and 2 crossover points, respectively. Thus, for PX65/JX1, the strain energy is ~3 kcal/mol/bp, while it is ~13 kcal/mol/bp for JX2, JX3 and JX4. Another measure of the stability is the change in the structure from the minimum energy structure to the equilibrium structure at 300 K, denoted as root-mean-square deviation in coordinates (CRMSD). We find that CRMSD is ~3.5 Å for PX65, increases to 6 Å for JX1 and increases to 10 Å for JX2/JX3/JX4. As the number of crossover points decreases, the distance between the two double helical domains of the PX/JX molecules increases from ~20 Å for PX65 to 23 Å for JX4. This indicates that JX2, JX3 and JX4 are less likely to form, at least in with Na⁺. However, in all the cases, the two double helical domains have average helicoidal parameters similar to a typical B-DNA of similar length and base sequence.     

The stereochemistry of a four-way DNA junction: a theoretical study.

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60 degrees. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60 degrees rather than 120 degrees), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.     

T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence.

Endonuclease VII of phage T4 resolves Holliday structures in vitro by nicking pairs of strands across the junction. We report here analyses of this reaction between endonuclease VII and a Holliday structure analogue, made in vitro from synthetic oligonucleotides. The enzyme cleaves the structure in a non-concerted way and nicks each strand independently. Combinations of nicks with counter-nicks in strands across the junction resolve the construct. The specificity of the enzyme for DNA secondary structures was tested with a series of branched molecules made from oligonucleotides with the same nucleotide sequence in one strand. Results show that the number, location and relative cleavage efficiencies depend largely on the local nucleotide sequence, rather than on the branch type. In particular, endonuclease VII cleaves a complete four-armed cruciform as efficiently as a three-armed Y-junction or its derivatives, a semi-Y, a fork with two single-strand overhangs, a single-strand overhang, and a nicked DNA. However, exchange or addition of one or more nucleotides within the cleavage area flanking the structural signal for endonuclease VII strongly affects the cleavage pattern as well as their relative efficiency of usage. Examples with a single-stranded overhang are presented and show in summary that the enzyme has a fivefold preference for pyrimidines rather than purines.