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.     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Experimentally derived nearest-neighbor parameters for the stability of RNA three- and four-way multibranch loops.

Algorithms for predicting RNA secondary structure require approximations for the free energies of multibranch loops, also called junctions. The stabilities of 62 RNA duplexes with three- and four-way multibranch loops were determined by optical melting. To account for the observed sequence dependence, a revised loop free-energy approximation is proposed that accounts for the strain in three-way junctions with fewer than two unpaired nucleotides, penalizes asymmetry in the distribution of unpaired nucleotides, and gives a bonus for four-way loops relative to three-way loops. Parameters for this equation were determined by linear regression.     

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.     

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.     

Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme

Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.     

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 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.     

Construction of a DNA four-way junction: Design and NMR spectroscopy

In 1964 Holliday postulated the formation of cruciform structures (four-way junctions) in duplex DNA as intermediate in genetic recombination. Since then, many biochemical and biophysical investigations were directed at solving questions concerning structural details of stable four-way junctions. Thus far, NMR spectroscopy played a minor part in these investigations on account of the minimum size of the molecule (expressed as the number of nucleotide residues) that was thought necessary to produce a stable cruciform structure. Indeed, the smallest four-way junction studied thus far by NMR methods was built from four separate DNA strands, each containing 16 nucleotides, a total of 64. Obviously, with such a large structure one runs into assignment problems. We considered the possibility of constructing a stable four-way junction from a single strand of DNA. The underlying idea was to make use of our detailed knowledge of the building principles of stable minihairpin loops. These loops, containing only two nucleotides to bridge the gap between antiparallel strands, are maximally stable in DNA sequences like 5-d(-C-TT-G-), where C and G form a normal Watson-Crick base pair and the two T residues cross the minor groove to form the minihairpin loop. Three of such miniloops could in principle cap three arms of the cruciform. The fourth arm would have an open end. The problem to be solved is to find the minimum length that is required to insure stability of the three closed arms and of the fourth open arm. We were successful with a structure that has three short stems (four base pairs each) and an open-end stem consisting of eight base pairs, a total of 46 residues. NMR experiments, carried out on this molecule in the presence of Mg²⁺, showed details of folding which have never been observed before.     

A four-way junction with triple-helical arms: design, characterization, and stability

The formation of the four-way junction containing four triple-helical arms has been demonstrated using chemical methods (polyacrylamide gel electrophoresis and chemical footprinting using OsO(4) as a probe) and physical methods (UV absorbance melting and DSC). The junction J(T1T3) was assembled from two 20-mer purine strands and two 44-mer pyrimidine strands. To determine the contribution of the different arms to the stability of the complete structure of J(T1T3), the junction was compared to two simplified substructures, J(T1) and J(T3), respectively. Common to these complexes is the underlying double-helical four-way junction Js. Addition of Na(+) had a profound effect on stabilizing and subsequently folding the junctions into the stacked X-structures. The following results support the structure present: (i) The native polyacrylamide electrophoresis exhibits only a single band(s) corresponding to one species present when all four single strands are mixed in equal amounts. (ii) OsO(4) modifications were investigated at pH 5.0 and in the presence of 10 mM Mg(2+) and 100 mM Na(+). There is no cleavage of thymine residues at the branch point and throughout the structure. (iii) The thermal unfolding of J(T1) and J(T3) illustrates that the triple-helical arms are more stable than the double-helical arms which are contained in these junctions and that J(T1T3) with four triple-helical arms is slightly more stable than J(T1) and J(T3). (iv) The calorimetric transition enthalpies determined for the arms of J(T1T3) are comparable to those associated with the unfolding of its corresponding arms in J(T1) and J(T3). The results also illustrate that the formation of the junctions is not restricted by the pH, [Na(+)], sequence composition of the arms, and/or the loop position.     

Thermodynamic properties of an intramolecular DNA four-way junction

We have investigated the thermodynamic properties of two homologous DNA four-way junctions, J4 and J4M, based on 46-mer linear DNA molecules. J4 and J4M have the same base sequence with the only difference that the latter contains an uncharged methylene-acetal linkage, -O3'-CH2-O5', instead of the phosphodiester linkage, -O3'-PO2-O5'-, between the residues T18 and C19. The comparison of the thermal unfolding of the J4 junction and J4M junction serves to investigate the effect of the uncharged methylene-acetal linkage on the stability of the junction. Our analysis is based on CD, UV absorbance spectroscopy, DSC, and chemical footprinting. The aim is to characterize in detail the structure and stability of the junctions. As demonstrated before by NMR, in the presence of 5 mM MgCl2 +/- 50 mM NaCl, both J4 and J4M form a complete four-way junction. This is now evidenced by protection from OsO4 cleavage (chemical footprinting). We can assume that full base pairing occurs throughout the arms even at the center of the junction. CD spectra suggest that the helices within the junctions adopt the regular B-DNA conformation. Almost identical melting temperatures and unfolding enthalpies are obtained for J4 and J4M both by UV and DSC. Furthermore, the Van't Hoff enthalpy (DeltaHVH) derived from UV melting equals the calorimetric enthalpy (DeltaHcal), which means that the melting process of the structures proceeds in a two-state manner. All results taken together support the conclusion that there are no major conformational and energetic differences between J4 and J4M. The inclusion of the uncharged methylene-acetal group into the junction has no effect on its stability.     

The role of RuvA octamerization for RuvAB function in vitro and in vivo

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.     

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.     

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The T_m values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5ʹ→3ʹ (T5 Exo) and 3ʹ→5ʹ direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.     

The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1

Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression. Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs. Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1. Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system. These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding. However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes. We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.     

The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite.

The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite. Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis. Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere. A low level of modification was observed at the four-way junction of a cruciform. The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction.     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.     

Structural motifs in ribosomal RNAs: implications for RNA design and genomics

The various motifs of RNA molecules are closely related to their structural and functional properties. To better understand the nature and distributions of such structural motifs (i.e., paired and unpaired bases in stems, junctions, hairpin loops, bulges, and internal loops) and uncover characteristic features, we analyze the large 16S and 23S ribosomal RNAs of Escherichia coli. We find that the paired and unpaired bases in structural motifs have characteristic distribution shapes and ranges; for example, the frequency distribution of paired bases in stems declines linearly with the number of bases, whereas that for unpaired bases in junctions has a pronounced peak. Significantly, our survey reveals that the ratio of total (over the entire molecule) unpaired to paired bases (0.75) and the fraction of bases in stems (0.6), junctions (0.16), hairpin loops (0.12), and bulges/internal loops (0.12) are shared by 16S and 23S ribosomal RNAs, suggesting that natural RNAs may maintain certain proportions of bases in various motifs to ensure structural integrity. These findings may help in the design of novel RNAs and in the search (via constraints) for RNA-coding motifs in genomes, problems of intense current focus.     

Computational investigation of the impact of core sequence on immobile DNA four-way junction structure and dynamics

Immobile four-way junctions (4WJs) are core structural motifs employed in the design of programmed DNA assemblies. Understanding the impact of sequence on their equilibrium structure and flexibility is important to informing the design of complex DNA architectures. While core junction sequence is known to impact the preferences for the two possible isomeric states that junctions reside in, previous investigations have not quantified these preferences based on molecular-level interactions. Here, we use all-atom molecular dynamics simulations to investigate base-pair level structure and dynamics of four-way junctions, using the canonical Seeman J1 junction as a reference. Comparison of J1 with equivalent single-crossover topologies and isolated nicked duplexes reveal conformational impact of the double-crossover motif. We additionally contrast J1 with a second junction core sequence termed J24, with equal thermodynamic preference for each isomeric configuration. Analyses of the base-pair degrees of freedom for each system, free energy calculations, and reduced-coordinate sampling of the 4WJ isomers reveal the significant impact base sequence has on local structure, isomer bias, and global junction dynamics.     

Fluorescence competition and optical melting measurements of RNA three-way multibranch loops provide a revised model for thermodynamic parameters

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.