Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Pyrene-modified guanosine as fluorescent probe for DNA modulated by charge transfer

8-(Pyren-1-yl)-2'-deoxyguanosine (Py-G) was incorporated synthetically as a modified DNA base and optical probe into oligonucleotides. A variety of Py-G-modified DNA duplexes have been investigated by methods of optical spectroscopy. The DNA duplex hybridization can be observed by both fluorescence and absorption spectroscopy since the Py-G group exhibits altered properties in single strands versus double strands for both spectroscopy methods. The fluorescence enhancement upon DNA hybridization can be improved significantly by the presence of 7-deazaguanin as an additional modification and charge acceptor three bases away from the Py-G modification site. Moreover, Py-G in DNA can be applied as a photoinducable donor for charge transfer processes when indol is present as an artificial DNA base and charge acceptor. Correctly base-paired duplexes can be discriminated from mismatched ones by comparison of their fluorescence quenching.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.     

Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin

The 3,3'-diethylthiacarbocyanine (DiSC(2)(5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-beta-cyclodextrin (Succ-beta-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC(2)(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.     

DNA branch nuclease activity of vaccinia A22 resolvase

DNA replication, recombination, and repair can result in formation of diverse branched DNA structures. Many large DNA viruses are known to encode DNA branch nucleases, but several of the expected activities have not previously been found among poxvirus enzymes. Vaccinia encodes an enzyme, A22 resolvase, which is known to be active on four-stranded DNA junctions (Holliday junctions) or Holliday junction-like structures containing three of the four strands. Here we report that A22 resolvase in fact has a much wider substrate specificity than previously appreciated. A22 resolvase cleaves Y-junctions, single-stranded DNA flaps, transitions from double strands to unpaired single strands ("splayed duplexes"), and DNA bulges in vitro. We also report site-directed mutagenesis studies of candidate active site residues. The results identify the likely active site and support a model in which a single active site is responsible for cleavage on Holliday junctions and splayed duplexes. Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.     

Evidence from CD spectra and melting temperatures for stable Hoogsteen-paired oligomer duplexes derived from DNA and hybrid triplexes.

The pyr*pur.pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crick-paired with an antiparallel pyrimidine strand (pur.pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paired duplexes has been reported. Using oligomer triplexes of repeating d(AG)12 and d(CT)12 or r(CU)12 sequences that were 24 nt long, we found that hybrid RNA*DNA as well as DNA*DNA Hoogsteen-paired strands of triplexes can be more stable than the Watson-Crick-paired strands at low pH. The structures and relative stabilities of these duplexes and triplexes were evaluated by circular dichroism (CD) spectroscopy and UV absorption melting studies of triplexes as a function of pH. The CD contributions of Hoogsteen-paired RNA*DNA and DNA*DNA duplexes were found to dominate the CD spectra of the corresponding pyr*pur.pyr triplexes.     

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.     

Fast and easy colorimetric tests for single mismatch recognition by PNA–DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-β-cyclodextrin

The 3,3′-diethylthiacarbocyanine (DiSC₂(5)) dye is able to aggregate on full matched PNA–DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-β-cyclodextrin (Succ-β-CyD) to the solutions containing PNA–DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC₂(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.     

Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes

Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.     

Chemical reactions in double-stranded nucleic acids. III. Synthesis of terminal inverted repeats of the IS1 element

Three 35 bp-DNA duplexes have been assembled from synthetic oligonucleotides by means of DNA ligase or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in two parallel series of experiments. The "top" strands of these duplexes correspond either to the imperfect (natural) or perfect terminal inverted repeats of the IS1. Tm of DNA duplexes composed of 2 to 6 different oligonucleotides were investigated by UV spectroscopy. It was shown that DNA ligase effectively joined oligonucleotides even under conditions of DNA duplex instability. However, there is a minimum duplex size (within the range of 9-15 bp) below which the enzymatic ligation is ineffective. Chemical assembly of duplexes took place only if the double helix was stable. The yield was 50% after two successive ligation cycles. Efficiency of the chemical ligation depends on the nature of the nucleotide units to be joined.     

Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme.

The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA, RNA hybrid duplexes and DNA-DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.     

Two strands of DNA are not equivalent as probes in hybridizations at low stringency

Only one of the two complementary strands of a restriction fragment hybridizes under low stringency conditions to a cloned Arabidopsis thaliana genomic DNA fragment. We propose that this effect is caused by the energetic nonequivalence of the two possible mismatched duplexes, resulting from the accumulation of mismatches and extrahelical bases. These mismatches will differ between the two duplexes. The choice of probe strand may therefore be important for the success of heterologous hybridizations utilizing single-stranded probes.     

Prediction of hybridization and melting for double-stranded nucleic acids

This article presents a general statistical mechanical approach to describe self-folding together with the hybridization between a pair of finite length DNA or RNA molecules. The model takes into account the entire ensemble of single- and double-stranded species in solution and their mole fractions at different temperatures. The folding and hybridization models deal with matched pairs, mismatches, symmetric and asymmetric interior loops, bulges, and single-base stacking that might exist at duplex ends or at the ends of helices. All possible conformations of the single- and double-stranded species are explored. Only intermolecular basepairs are considered in duplexes at this stage.In particular we focus on the role of stacking between neighboring nucleotide residues of single unfolded strands as an important source of enthalpy change on helix formation which has not been modeled computationally thus far. Changes in the states of the single strands with temperature are shown to lead to a larger heat effect at higher temperature. An important consequence of this is that predictions of enthalpies, which are based on databases of nearest-neighbor energy parameters determined for molecules or duplexes with lower melting temperatures compared with the melting temperatures of the oligos for which they are used as a predictive tool, will be underestimated.     

Structural studies of heterochiral DNA/DNA, RNA/RNA, and DNA/RNA duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

DNA hybrids stabilized by heterologies.

The double D-loop DNA hybrid contains four DNA strands following hybridization of two RecA protein coated complementary single-stranded DNA probes with a homologous region of a double-stranded DNA target. A remarkable feature of the double D-loop DNA hybrids is their kinetic stabilities at internal sites within linear DNA targets after removal of RecA protein from hybrids. We report here that heterologous DNA inserts in one or both probe strands affect the kinetic stability of protein-free double D-loop hybrids. DNA heterologies normally distort DNA-DNA hybrids and consequently accelerate hybrid dissociation. In contrast, heterologous DNA inserts impede dissociation of double D-loops, especially when the insert sequences interact with each other by DNA base pairing. We propose a mechanism for this kinetic stabilization by heterologous DNA inserts based on the hypothesis that the main pathway of dissociation of double D-loop DNA hybrids is a DNA branch migration process involving the rotation of both probe-target duplexes in the hybrids. Heterologous DNA inserts constrain rotation of probe-target duplexes and consequently impede hybrid dissociation. Potential applications of the stabilized double D-loops for gene targeting are discussed.