Asymmetric structure of a three-arm DNA junction

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)     

The three-way DNA junction is a Y-shaped molecule in which there is no helix-helix stacking.

We have studied the structure of a number of three-way DNA junctions that were closely related in sequence to four-way junctions studied previously. We observe that the electrophoretic mobility of the species derived by selective shortening of one arm of a junction are very similar whichever arm is shortened, and that this remains so whether or not magnesium is present in the buffer. This suggests that the angles subtended between the arms of the three-way junctions are similar. All thymine bases located immediately at the junction are reactive to osmium tetroxide, indicating that out-of-plane attack is not prevented by helix-helix stacking, and this is also independent of the presence or absence of metal cations. The results suggest that the three-way junction cannot undergo an ion-induced conformational folding involving helical stacking, but remains fixed in a Y-shaped extended conformation. Thus the three- and four-way junctions are quite different in character in the presence of cations.     

DNA recombination: holliday junctions dynamics and branch migration

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.     

Gel electrophoretic analysis of the geometry of a DNA four-way junction

Branched DNA molecules (Holliday structures) are believed to be key intermediates in the process of homologous genetic recombination. However, despite the importance of such structures, their transient nature makes it difficult to analyze their physical properties. In an effort to evaluate several models for the geometry of such branched molecules, a stable, synthetic DNA four-way junction has been constructed. The geometry of the synthetic junction has been probed by gel electrophoresis, utilizing the fact that bent DNA molecules demonstrate reduced mobilities on polyacrylamide gels to an extent that varies with the degree of the bend angle. From the synthetic four-way junction, we have produced a set of molecules in which all combinations of two junction arms have been extended by 105 base-pairs. The electrophoretic mobilities of the extended junctions differ in a manner which indicates that the junction is not a completely flexible structure; nor is it tetrahedral or planar-tetragonal. Instead, the four strands that comprise the DNA four-way junction are structurally non-equivalent. The significance of these observations with regard to previous models for four-way junction geometry is discussed.     

Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch

Four-arm DNA branched junctions are stable analogues of Holliday recombinational intermediates. A number of four-arm DNA junctions synthesized from oligonucleotides have now been studied. Gel mobility or chemical footprinting experiments on several immobile four-arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other. We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference. The pattern of self-protection of the strands of J1c exposed to Fe(II).EDTA-induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over. Moreover, the Fe(II).EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1. The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4 degrees C and in the presence of 10 mM Mg2+, based on chemical reactivity data. This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7. This difference could reflect either sequence-dependent differences in the equilibrium among isomers, or in the structure of these junctions. Chemical footprinting experiments using the probes MPE.Fe(II) and (OP)2Cu(I) show that the high-affinity ligand binding site in immobile junctions is determined by junction geometry.     

The role of metal ions in the conformation of the four-way DNA junction.

Metal ions fold DNA junctions into a compact conformation that confers protection of all thymine bases to modification by osmium tetroxide. In the absence of the cation the arms of the junction are fully extended in an approximately square-planar configuration. Group IIa cations are effective in achieving a folded conformation of the junction at 80-100 microM, and there is an excellent agreement between the ionic concentrations that fold the junctions as deduced from gel electrophoretic experiments, and those that prevent osmium tetroxide reaction at the junction. Hexamminecobalt(III) achieves full folding at 2 microM, while spermine and spermidine are effective at 25 microM. Some transition metal ions such as Ni(II) may replace the group IIA cations. Monovalent ions of group IA are only partially effective in folding the junctions. Very much higher concentrations are necessary, gel electrophoretic mobilities suggest that a less symmetrical conformation is adopted and thymine bases at the junction remain reactive to osmium tetroxide. Charge-charge interactions at the centre of the junction are structurally extremely important. Substitution of junction phosphate groups by uncharged methyl phosphonates severely perturbs the structure of the junction. If just two phosphates are substituted, diametrically facing across the junction, the structure always folds in order to place the electrically neutral phosphate on the exchanging strands. We suggest that folding of the junction into the stacked X-structure generates electronegative clefts that can selectively bind metal ions, depending on the chemistry, size and charge of the ion. Moreover, occupation of these cavities is essential for junction folding, in order to reduce electrostatic repulsion.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.     

Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.     

Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs.

We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.     

The dynamic nature of the four-way junction of the hepatitis C virus IRES

Translation is initiated within the RNA of the hepatitis C virus at the internal ribosome entry site (IRES). The IRES is a 341-nucleotide element that contains a four-way helical junction (IIIabc) as a functionally important element of the secondary structure. The junction has three additional, nonpaired nucleotides at the point of strand exchange on one diagonal. We have studied the global conformation and folding of this junction in solution, using comparative gel electrophoresis and steady-state and time-resolved fluorescence resonance energy transfer. In the absence of divalent metal ions, the junction adopts an extended-square structure, in contrast to perfect four-way RNA junctions, which retain coaxial helical stacking under all conditions. The IIIabc junction is induced to fold on addition of Mg(2+), by pairwise coaxial stacking of arms, into the conformer in which the unpaired bases are located on the exchanging strands. Fluorescence lifetime measurements indicate that in the presence of Mg(2+) ions, the IIIabc junction exists in a dynamic equilibrium comprising approximately equal populations of antiparallel and parallel species. These dynamic properties may be important in mediating interactions between the IRES and the ribosome and initiation factors.     

Effects of polyvalent cations on the folding of an rRNA three-way junction and binding of ribosomal protein S15.

The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA. Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels. Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction. NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar. Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure. The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (k(on)) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.     

The structure of the Holliday junction, and its resolution

The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.     

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.     

Characterization of a bimobile DNA junction

We present here a chemical and enzymatic footprinting analysis of a branched DNA molecule formed from four complementary 50-mer strands. These strands are designed to form a stable junction, in which two steps of branch point migration freedom are possible. Exposure of the junction to Fe(II).EDTA shows protection of 3 or 4 residues in each strand at the branch, while two resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7), cleave all four strand near the branch. Chemical footprinting of this junction using the reagents MPE.Fe(II) and (OP)2Cu(I) shows that the branch site is hyper-reactive to cutting induced by these probes as it is in an immobile four-arm junction. The effects involve more residues than in the immobile case. In the absence of divalent cations, the structure of the junction alters, sites of enhanced cleavage by MPE.Fe(II) and (OP)2Cu(I) disappear, and purines at the branch become reactive to diethyl pyrocarbonate. Our interpretation of these results is based on the properties of immobile junction analogs and their response to these probes. In the presence of Mg2+, the three migrational isomers coexist, each probably in the form of a 2-fold symmetric structure with two helical arms stacked.     

Parallel helical domains in DNA branched junctions containing 5',5' and 3',3' linkages

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms that flank a branch point. In the presence of Mg2+, the four arms are known to stack in pairs forming two helical domains whose orientations are antiparallel but twisted by about 60 degrees. The basis for the antiparallel orientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these two possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the branch point, the strand opposite it contains a 5',5' linkage, and the other two strands contain conventional 3',5' linkages. Electrostatic effects are expected to lead to an antiparallel structure in this system. We have characterized the molecule in comparison with a conventional immobile branched junction by Ferguson analysis and by observing its thermal transition profile; the two molecules behave virtually identically in these assays. Hydroxyl radical autofootprinting has been used to establish that the unusual linkages occur at the branch point and that the arms stack to form the same domains as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. Remarkably, we find them to be closer to parallel than to antiparallel, suggesting that the preferred structure of the branch point dominates over electrostatic repulsion. We have controlled for the number of available bonds in the branch point, for gel concentration, and for the role of divalent cations. This finding suggests that control of branch point structure alone can lead to parallel domains, which are generally consistent with recombination models derived from genetic data.     

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.