A kinetic model for the B–Z transition of poly[d(G-C)]·poly[d(G-C)] and poly[d(G-m5C)]·poly[d(G-m5C)]

The analysis of the kinetic data of the B-Z conformational changes induced by salt in sized double-stranded poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)] polymers indicated that there exists a salt threshold which reveals some largely, as yet, unrecognised characteristics of the transition. It was observed that there is a direct correlation between the length of the polymer and the rate of the B-Z transition when the salt concentration in the polymer solution is lower than the salt threshold. The correlation is inverse when the salt concentration is higher than the salt threshold. Thus, the molecular mechanism of the B- to Z-DNA transition varies depending on whether the salt concentration is higher or lower than the threshold. In this context, we have found that the contrasting results reported in the literature describing the rate of the B-Z transition are not contradictory but complementary. The finding of a salt threshold leads to the establishment of a relationship between the cooperativity index of the B-Z transition and the polymer chain length. That relationship is dependent on the chemical structure of the polymer but is temperature independent.     

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

Kinetics of the salt-induced B- to Z-DNA transition

The salt-induced B- to Z-DNA conformational transition is a cooperative- and time-dependent process. From a modified form of the logistic equation which describes an equilibrium between two states we have deduced a kinetic function to quantify the degree of the B to Z transition of a synthetic (dG-dC) ⋅ (dG-dC) polynucleotide. This function was obtained by introduction of time as a variable in the logistic function so that the equilibrium constant, K, is replaced by a new constant K _s , characteristic of the type of salt used. This constant is defined as the salt concentration needed to reach the B-Z transition-midpoint in the time unit. The equation fits the data obtained by circular dichroism (CD) for changes in molecular ellipticity of poly(dG-m⁵dC) ⋅ poly(dG-m⁵dC) and poly(dG-dC) ⋅ poly(dG-dC) incubated with various concentrations of mono-, di-, and trivalent salts at a constant temperature. The derived expression may be a very useful tool for studying the kinetics of the B- to Z-DNA transition. Received: 1 December 1997 / Revised version: 16 March 1998 / Accepted: 27 March 1998     

B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

Left-handed Z Form in Superhelical DNA: A Theoretical Study

Abstract

This is a comprehensive statistical mechanical treatment of the Z form formation in purine- pyrimidine stretches of different length inserted into superhelical DNA. The B-Z transition for short inserts is shown to follow the “all-or-none” principle. Over some critical value of the insert length n, the B-Z transition in the insert proceeds in two stages. The flipping of m base pairs into the Z form is followed by a gradual growth of the Z-form stretch until it occupies the whole insert. By fitting the theoretical transition curves to experimental ones the fundamental thermodynamic parameters of the B-Z transition have been determined: the B-Z junction energy Fj = 4–5kcal?mol^?1 and the free energy change ΔF_B-Z = 0.5–0.7 kcal?mol^?1 under standard salt conditions. Calculations show that the B-Z transition in short purine-pyrimidine inserts may be seriously affected by cruciform formation in the carrier DNA.     

B,Z conformations and mechanism of the Z-B-coil transitions of the self-complementary deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD spectroscopy

The helical structures of d(C-G-m5C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and 1H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (greater than 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration). The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95 degrees C) only the coil form (S) is present. Below 55 degrees C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m5C-G-C-G-m5C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z in equilibrium B in equilibrium S. The present data show that even at high salt concentration where only the Z form of d(C-G-m5C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

B-Z Cooperativity and Kinetics of Poly(dG-m5dC) are Controlled by an Unfavorable B-Z Interface Energy

Abstract

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m⁵dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl₂, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl, in 50 mM Nad, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 ± 0.3 kcal/mol and is length independent Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 ± 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Salt Dependence of DNA Structural Stabilities in Solution. Theoretical Predictions Versus Experiments

Abstract

The predictions of currently available theories for treating DNA-diffuse ionic cloud free energy contributions to conformational stability have been tested against experimental data for salt induced B-Z and B-A transitions. The theories considered are (i) Manning's counterion condensation approach (CC), (ii) the idealized Poisson-Boltzmann approximation (PB), and (iii) the potentials of mean force (PMF) approach proposed by Soumpasis. As far as we can judge from comparison with the set of experimental data currently available, it is found that only the latter theory yields satisfactory quantitative results for the dependence of the B-Z and B-A relative stabilities on monovalent salt concentration. The correct application of the PB and CC theories does not yield very low salt Z-B transitions, in contradiction to earlier assertions. At low salt concentrations the PB theory is qualitatively correct in predicting that the B form is electrostatically more favorable than both the A and B forms, whereas the CC theory is qualitatively wrong predicting that Z-DNA is more stable than both B and A DNA.     

Stochastic distribution of a short region of Z-DNA within a long repeated sequence in negatively supercoiled plasmids

We have analyzed, at nucleotide resolution, the progress of the B-to-Z transition as a function of superhelical density in a 2.2-kilobase plasmid containing the sequence d(C-A)31.d(T-G)31. The transition was monitored by means of reactivity to two chemical probes: diethyl pyrocarbonate, which is sensitive to the presence of Z-DNA, and hydroxylamine, which detects B-Z junctions. At a threshold negative superhelical density between about 0.048 and 0.056, hyper-reactivity to diethyl pyrocarbonate appears throughout the CA/TG repeat and remains as the superhelical density is further increased. However, there is no reactivity characteristic of B-Z junctions until the superhelical density reaches 0.084, when single cytosines at each end of the repeat become hyper-reactive to hydroxylamine. A two-dimensional gel analysis of this system by others (Haniford, D. B., and Pulleyblank, D. E. (1983) Nature 302, 632-634) indicates that only about half of the 62 base pairs of the CA/TG repeat undergo the initial transition at omega = 0.056. Our results indicate that this region of Z-DNA is free to exist anywhere along the CA/TG repeat and is probably constantly in motion. Well defined B-Z junctions are seen only when there is sufficient supercoiling to convert the entire CA/TG sequence to Z-DNA. The implications for possible B-Z transitions in chromosomal domains of different sizes are discussed.     

Salt dependence of DNA structural stabilities in solution. Theoretical predictions versus experiments

The predictions of currently available theories for treating DNA-diffuse ionic cloud free energy contributions to conformational stability have been tested against experimental data for salt induced B-Z and B-A transitions. The theories considered are (i) Manning's counterion condensation approach (CC), (ii) the idealized Poisson-Boltzmann approximation (PB), and (iii) the potentials of mean force (PMF) approach proposed by Soumpasis. As far as we can judge from comparison with the set of experimental data currently available, it is found that only the latter theory yields satisfactory quantitative results for the dependence of the B-Z and B-A relative stabilities on monovalent salt concentration. The correct application of the PB and CC theories does not yield very low salt Z-B transitions, in contradiction to earlier assertions. At low salt concentrations the PB theory is qualitatively correct in predicting that the B form is electrostatically more favorable than both the A and Z forms, whereas the CC theory is qualitatively wrong predicting that Z-DNA is more stable than both B and A DNA.     

Effect of basic oligopeptides on the B-Z transition of poly(dG-dC). poly(dG-dC) in water-methanol solutions

The effect of basic oligopeptides (Lys-Ala-Ala)n (n = 1-5, 10) and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-dC).poly(dG-dC) in water-methanol solutions was investigated using CD and uv spectroscopy. In the absence of peptides, the concentration of methanol at the midpoint of the B-Z transition is 64% at 25 degrees C. The transition is temperature dependent and the B conformation is preferred at higher temperatures. All peptides tested shift the midpoint of the B-Z transition to lower concentrations of methanol. For shorter peptides this effect increases with an increasing number of monomeric units, showing the importance of the number of positive charges in the peptide molecule. Al conditions of low methanol content, the trimer and tetramer of the (Lys-Leu-Ala)n series have a greater effect on the B-Z transition than the corresponding oligomers of the (Lys-Ala-Ala)n series. This indicates an important influence of the presence of hydrophobic groups in the peptide side chains on the binding. In the presence of peptides, the B-Z transition is also temperature dependent and the B conformation is preferred at higher temperatures. The addition of peptides results in an increase of the transition midpoint and of the transition width. These parameters were used for the calculation of the transition enthalpy delta HB-Z in 65% methanol, which is -1.15 +/- 0.25 kcal/base pair. Since the van't Hoff enthalpy delta HVH calculated from the temperature dependence of the B-Z transition in the absence of peptides is -130 kcal/mol, the length of the cooperative unit is about 110 base pairs. The results suggest that the mechanism of Z-DNA induction is similar but not identical with that involved in the action of metal cations in aqueous solution.     

A Model of the Strain-induced B-Z Transition

Abstract

The B-to-Z transition in supercoiled circular DNA is modeled as a strain-induced nonlinear excitation process. Using a model, in which DNA is regarded as a chain of units with a bistable energy function along the twisting coordinate together with a harmonic inter-unit interaction, we show that a Z region and the accompanying two B-Z junctions of finite width appear naturally as a solution of nonlinear equations, when the strain exceeds a critical value. We examine the B-Z transition behaviour as a function of twist under various situations. We also analyse available experimental results on B-Z transition in supercoiled plasmid with G-C insertions by this mechanistic model in order to estimate the magnitude of model parameters. The energy barrier of the B-Z transition is estimated to be of the order of 1 kcal/mole per base pair. The analysis shows that if the length of the insertion is less than a certain value, the entire insertion converts to Z form at a transition point, but if the insertion is much longer, the B-Z transition exhibits a different behavior, in which part of the insertion flips to Z form and the Z region expands linearly upon changing linking number.     

Left-handed Z form in superhelical DNA: a theoretical study

This is a comprehensive statistical mechanical treatment of the Z form formation in purinepyrimidine stretches of different length inserted into superhelical DNA. The B-Z transition for short inserts is shown to follow the "all-or-none" principle. Over some critical value of the insert length n, the B-Z transition in the insert proceeds in two stages. The flipping of m base pairs into the Z form is followed by a gradual growth of the Z-form stretch until it occupies the whole insert. By fitting the theoretical transition curves to experimental ones the fundamental thermodynamic parameters of the B-Z transition have been determined: the B-Z junction energy Fj = 4-5kcal.mol-1 and the free energy change delta FBZ = 0.5-7.0 kcal.mol-1 under standard salt conditions. Calculations show that the B-Z transition in short purinepyrimidine inserts may be seriously affected by cruciform formation in the carrier DNA.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

During B-Z Transition There is No Large Scale Breakage of Watson-Crick Base Pairs A Direct Demonstration Using 500 MHz 1H NMR Spectroscopy

Abstract

Monitoring of the Watson-Crick GNH1 proton in poly(dG-dC)-poly(dG-dC) at 500 MHz in 90% H₂0:10% D₂o at 30° C as a function of NaCl concentration (1.5 to 3.6 M), demonstrates that the bases retain Watson-Crick pairing throughout the transition. This observation unequivocally demonstrates that during the B-Z transition there is no large scale and detectable base pair opening and that macroscopically the phenomenon can be described as a direct helix to helix transition. We present frame by frame, an energetically sound stereodynamical trajectory for this transfiguration from right-handed B-DNA to left-handed Z-DNA.     

B-Z cooperativity and kinetics of poly(dG-m5dC) are controlled by an unfavorable B-Z interface energy

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m5dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl2, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl2 in 50 mM NaCl, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 +/- 0.3 kcal/mol and is length independent. Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 +/- 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event. Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Studies on the Critical Micellar Concentration and Phase Transitions of Stearoylcarnitine

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 μM at pH 3.0 to ca. 25 μM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 μM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5°C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (T _t) shifts to ca. 50.5°C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1°C at pH 3.0, which increased with increasing pH up to a value of 55.3°C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and T _t suggest that the pK _a of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes).     

The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

B to Z transitions of the short DNA hairpins formed from the oligomer sequences: d[(CG)3X4(CG)3] (X = A, T, G, C).

Circular Dichroism (CD) spectra were collected as a function of sodium perchlorate concentration [NaClO4] for the set of DNA hairpins formed from the oligomer sequences d[(CG)3X4(CG)3] where X = A, T, G or C. Over the range in salt concentration from 0 to 4.0 M NaClO4, the CD spectra invert in a manner characteristic of the B to Z transition. A factor analysis routine is described and employed to determine the least number of basis spectra required to fit the measured spectra of each hairpin over the entire salt range examined. In every case, linear combinations of only two sub-spectra fit the experimental spectra of the hairpins with greater than 98% accuracy, indicating the spectrally monitored structural transitions are two-state. From the relative weights of the individual sub-spectra, B-Z transition curves are constructed. The transitions are analyzed in terms of a simple two-state equilibrium model which yields an evaluation of the transition free-energy, delta GB-Z, as a function of NaClO4 concentration. At 1.0 M NaClO4 and 21 degrees C, delta GB-Z = 5.4, 4.9, 3.6 and 2.3 kcal/mole for the G4, T4, A4 and C4 loop hairpins, respectively.