Stability and structure of three-way DNA junctions containing unpaired nucleotides.

Non-paired nucleotides stabilize the formation of three-way helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative three-way junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that delta delta G degrees (37 degrees C) = +0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Y-shaped, rather than a T-shaped junction.     

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.     

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.     

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

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.     

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.     

Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold

Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.     

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.     

Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection

The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.     

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)     

Structures of helical junctions in nucleic acids

Our knowledge of the architectural principles of nucleic acid junctions has seen significant recent advances. The conformation of DNA junctions is now well understood, and this provides a new basis for the analysis of important structural elements in RNA. The most significant new data have come from X-ray crystallography of four-way DNA junctions; incidentally showing the great importance of serendipity in science, since none of the three groups had deliberately set out to crystallise a junction. Fortunately the results confirm, and of course extend, the earlier conformational studies of DNA junctions in almost every detail. This is important, because it means that these methods can be applied with greater confidence to new systems, especially in RNA. Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly. Molecular modelling in conjunction with experiments is also a very important component of the general approach. Ultimately crystallography provides the gold standard for structural analysis, but the other, simple approaches have considerable value along the way. At the beginning of this review I suggested two simple folding principles for branched nucleic acids, and it is instructive to review these in the light of recent data. In brief, these were the tendency for pairwise coaxial stacking of helical arms, and the importance of metal ion interactions in the induction of folding. We see that both are important in a wide range of systems, both in DNA and RNA. The premier example is the four-way DNA junction, which undergoes metal ion-induced folding into the stacked X-structure that is based on coaxial stacking of arms. As in many systems, there are two alternative ways to achieve this depending on the choice of stacking partners. Recent data reveal that both forms often exist in a dynamic equilibrium, and that the relative stability of the two conformers depends upon base sequence extending a significant distance from the junction. The three-way junction has provided a good test of the folding principles. Perfect three-way (3H) DNA junctions seem to defy these principles in that they appear reluctant to undergo coaxial stacking of arms, and exhibit little change in conformation with addition of metal ions. Modelling suggests that such a junction is stereochemically constrained in an extended conformation. However, upon inclusion of a few additional base pairs at the centre (to create a 3HS2 junction for example) the additional stereochemical flexibility allows two arms to undergo coaxial stacking. Such a junction exhibits all the properties consistent with the general folding principles, with ion-induced folding into a form based on pairwise coaxial stacking of arms in one of two different conformers. The three-way junction is therefore very much the exception that proves the rule. It is instructive to compare the folding of corresponding species in DNA and RNA, where we find both similarities and differences. The RNA four-way junction can adopt a structure that is globally similar to the stacked X-structure (Duckett et al. 1995a), and the crystal structure of the DNAzyme shows that the stacked X-conformation can include one helical pair in the A-conformation (Nowakowski et al. 1999). However, modelling suggests that the juxtaposition of strands and grooves will be less satisfactory in RNA, and the higher magnesium ion concentration required to fold the RNA junction indicates a lower stability of the antiparallel form. Perhaps the biggest difference between the properties of the DNA and RNA four-way junctions is the lack of an unstacked structure at low salt concentrations for the RNA species. This clearly reflects a major difference in the electrostatic interactions in the RNA junction. In general the folding of branched DNA provides some good indications on the likely folding of the corresponding RNA species, but caution is required in making the extrapolation because the two polymers are significantly different. A number of studies point to the flexibility and malleability of branched nucleic acids, and this turns out to have particular significance in their interactions with proteins. Proteins such as the DNA junction-resolving enzymes exhibit considerable selectivity for the structure of their substrates, which is still not understood at a molecular level. Despite this, it appears to be universally true that these proteins distort the global, and in some cases at least the local, structure of the junctions. The somewhat perplexing result is that the proteins appear to distort the very property that they recognise. In general it seems that four-way DNA junctions are opened to one extent or another by interaction with proteins. (ABSTRACT TRUNCATED)     

Sequence-dependent folding of DNA three-way junctions

Three-way DNA junctions can adopt several different conformers, which differ in the coaxial stacking of the arms. These structural variants are often dominated by one conformer, which is determined by the DNA sequence. In this study we have compared several three-way DNA junctions in order to assess how the arrangement of bases around the branch point affects the conformer distribution. The results show that rearranging the different arms, while retaining their base sequences, can affect the conformer distribution. In some instances this generates a structure that appears to contain parallel coaxially stacked helices rather than the usual anti-parallel arrangement. Although the conformer equilibrium can be affected by the order of purines and pyrimidines around the branch point, this is not sufficient to predict the conformer distribution. We find that the folding of three-way junctions can be separated into two groups of dinucleotide steps. These two groups show distinctive stacking properties in B-DNA, suggesting there is a correlation between B-DNA stacking and coaxial stacking in DNA junctions.     

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.     

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (60° ) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (± 20° ) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.     

Quasi-equivalence in site-specific recombinase structure and function: crystal structure and activity of trimeric Cre recombinase bound to a three-way Lox DNA junction

The crystal structure of a novel Cre-Lox synapse was solved using phases from multiple isomorphous replacement and anomalous scattering, and refined to 2.05 A resolution. In this complex, a symmetric protein trimer is bound to a Y-shaped three-way DNA junction, a marked departure from the pseudo-4-fold symmetrical tetramer associated with Cre-mediated LoxP recombination. The three-way DNA junction was accommodated by a simple kink without significant distortion of the adjoining DNA duplexes. Although the mean angle between DNA arms in the Y and X structures was similar, adjacent Cre trimer subunits rotated 29 degrees relative to those in the tetramers. This rotation was accommodated at the protein-protein and DNA-DNA interfaces by interactions that are "quasi-equivalent" to those in the tetramer, analogous to packing differences of chemically identical viral subunits at non-equivalent positions in icosahedral capsids. This structural quasi-equivalence extends to function as Cre can bind to, cleave and perform strand transfer with a three-way Lox substrate. The structure explains the dual recognition of three and four-way junctions by site-specific recombinases as being due to shared structural features between the differently branched substrates and plasticity of the protein-protein interfaces. To our knowledge, this is the first direct demonstration of quasi-equivalence in both the assembly and function of an oligomeric enzyme.     

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.     

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.     

Structural recognition between a four-way DNA junction and a resolving enzyme

Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA-protein contacts in the arms containing the 5' ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I-junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands.