On the possibility of true phase transition in heterogeneous DNA during thermal denaturation under conditions close to equal stability of A+T and G+C pairs

The DNA helix–coil transition has been studied in the presence of high concentrations of manganese ions (about 10^?3M), which corresponds to the conditions close to equal stability of the A+T and G+C pairs, at the ionic strengths of 10^?1, 10^?2, and 1.6 × 10^?3M Na⁺. With the Mn²⁺ ion effect, the transition range is significantly reduced to not more than 0.2°C at 1.2 × 10^?3M Mn²⁺ and 1.6 × 10^?3M Na⁺. The melting curves display a sharp kink at the end of the helix–coil transition, which is interpreted as an indication of the second-order phase transition. It is shown that the melting curves obtained can be approximated by a simple analytical expression 1 – θ = exp[–a(t_c - t)], where θ is the DNA helix fraction, t_c is the phase transition temperature, and a is an empirical parameter characterizing the breadth of the melting range and responsible for the magnitude of a jump of the helicity derivative with respect to the temperature at the phase transition point.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Interactions des ions métalliques avec le DNA. IV. Fixation de i'ion cuivrique sur le DNA

The binding of cupric ion (Cu⁺⁺) to DNA was followed by spectrophotometry, melting profiles, and hydrodynamic techniques, in 0. 1M NaClO₄ and at pH 5. 6. A small amount of Cu⁺⁺ is bound specifically to bases (about 1 Cu⁺⁺ per 20 nucleotides), in agreement with polarographic and EPR data. A preferential stabilization of G–C pairs and only a slight increase of the flexibility of the molecule were observed. In 5 × 10^?3M NaClO₄, a higher number of nonhomogeneous binding sites is found by spectrophotometry. It is concluded that at least two types of sites are available for Cu⁺⁺. The first one, where Cu⁺⁺ is chelating N₇ of purines to phosphate, is observed only at low ionic strength and destabilizes the double helix. The second exists mainly at 0, 1M or higher ionic strength. All the sites are identical and could be attributed to two successive guanine residues in the same strand. Similar behavior was found for other divalent cations, e. g., Fe⁺⁺, Mn⁺⁺, and Co⁺⁺.     

Effects of calcium and manganese ions on the helix–coil transition of DNA

The thermal denaturation method was employed to study the effect of Ca²⁺ and Mn²⁺ ions on the DNA helix–coil transition parameters at Na⁺ concentrations of 10^?3–10^?1M. At low ion concentrations, thermal stability increases, the melting range passes through a maximum, and the denaturation curves become asymmetric. These changes are quantitatively similar for Mn²⁺ and Ca²⁺ ions. With a further increase in the concentration of bivalent ions, the conformational transition temperatures pass through a maximum, and the melting range first tends to saturation and then rapidly decreases to 1–2°C. The Mn²⁺ concentrations, at which the above effects occur, are an order of magnitude lower than the Ca²⁺ concentrations. Comparison of experimental results and calculation in terms of the ligand theory permitted estimation of binding constants characterizing association between Mn²⁺ and Ca²⁺ ions and bases of native and denatured DNA. We show that, unlike the interaction with phosphates, bivalent ion–DNA base binding is weakly dependent on monovalent ion concentration in the solution.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Effects of Na+ and Mg++ ions on the helix–coil transition of DNA

The effects of monovalent (Na⁺) and divalent (Mg⁺⁺) cations on the temperature and breadth of the helix–coil transition of phage DNA have been investigated. The experimental results confirm the findings of Dove and Davidson [J. Mol. Biol. 5 , 467–478 (1962)] for the limiting cases of zero divalent ion concentration and saturating levels of divalent ion, and extend their findings to the intermediate region of Mg⁺⁺ concentrations. A theory for the dependence of transition temperature on the ion concentrations is developed, utilizing the approach of Wyman [Adv. Protein Chem. 19 , 223–286 (1964)], modified to account for electrostatic nonideality of the polyelectrolytes. The theory is in agreement with Manning's treatment of the experiments of Dove and Davidson [Biopolymers 11 , 937–949, 951–955 (1972)] and is in fair agreement with experimental data over the entire range of ion concentrations. Further investigation of the structure and ion-binding properties of the denatured form will be required before a quantitative comparison between theory and experiment can be performed.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Effect of Mg++ and polyamines on proflavine binding to T2 DNA

Proflavine binding may be used as a probe of the environment and interactions of DNA. In this paper we report the effects of the divalent cations Mg⁺⁺ and putrescine and the trivalent cation spermidine on the proflavine–Na DNA binding equilibrium. Difference spectroscopy at 430 nm was used to determine apparent proflavine–DNA binding constants K at several concentrations of each cation for temperatures between 15 and 43°C, and at a constant total ionic strength of 0.1M. Mg⁺⁺, putrescine, and spermidine all have greater effects on K than expected on the basis of ionic strength alone in the order spermidine > Mg⁺⁺ ? putrescine. van't Hoff analysis of K(T) enabled calculation of ΔH° and ΔS°, which are affected differently by each cation. These differences are discussed qualitatively in terms of such concepts as release of condensed counterions, localized or unlocalized condensation, hydration, and restriction of molecular and internal rotation.     

Cooperative nonenzymic base recognition. Thermodynamics of the helix--coil transition of a monomer--polymer double helix

The double-helical complex formed from m⁶m⁹A and poly U has been characterized by circular dichroism and u.v. spectrophotometry. The circular dichroism of the complex is similar to that of the double-helical poly A poly U complex both in shape and in magnitude and thus indicates a quite similar structure. The double Helix–coil transition has been studied at various nucleotide concentrations and at three different ionic strengths. As expected for the binding of a base to a polymer, the Helix–coil transition is shifted to higher temperatures by increasing nucleotide concentrations, but is not affected by changes of the ionic strength. The melting curves are analyzed according to a linear Ising model taking the stacking of the monomers into account. At 0°C the equilibrium constant for nucleation is found to be 2–5 M^?1 and that for chain growth is 500 M^?1. The enthalpy change associated with chain growth is ?11.2 ± 1 kcal M^?1.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Salt-concentration dependence of melting profiles of lambda phage DNAs: evidence for long-range interactions and pronounced end effects.

Melting profiles of DNAs from wild-type λ phage and a deletion mutant phage λb2 were examined in a wide range of salt concentration. The fine structure of the melting profiles changed sharply with salt concentration, especially in the range [Na⁺] ? 10 mM. A comparison of the melting profiles between the wild-type and the deletion mutant DNAs provided good evidence for extremely high melting cooperativity under low salt conditions, which is clearly manifested as the long-range interactions and the pronounced end effects; a large melting peak appeared as a result of the b2 deletion without any inserted sequence in the salt range [Na⁺] ? 2.8 mM. It was also suggested that in the further reduced salt range [Na⁺] ? 2.0 mM, melting of a λ DNA molecule starts from its right end rather than the most (A + T)-rich central region. The molecular basis of the high melting cooperativity at low salt concentrations can be explained in terms of the increased free energy associated with loop formation in the double-helical structure of DNA.     

Adenosine Triphosphatases Bound to Plasmalemma, Mitochondria, and Microsomes or Present in the Cytoplasmic Phase from Potato Tubers

Between pH 4–10, basal ATPase activity, measured in the absence of mineral ions, was 10 to 100 times higher in the final cytoplasmic supernatant from potato tuber homogenates than in the membraneous fractions (purified plasmalemma, purified mitochondria and microsomes). The soluble ATPase was slightly inhibited, whereas the membrane-bound ATPases were all stimulated by Mg²⁺ ions. A further stimulation by Na⁺ or K⁺ ions was only observed in purified plasmalemma or mitochondria, at alkaline pH (7.5–9.5). At a fixed (Na⁺+ K⁺) concentrations (80 mM), this last stimulation was much greater in purified mitochondria (350%) than in plasmalemma (33%); it also increased with (Na⁺+ K⁺) concentrations up to 200 mM in mitochondria whereas, in plasmalemma, it was roughly constant for monovalent ion concentrations between 20 and 200 mM. General properties of the plasma membrane-bound ATPase have been determined, i.e. substrate specificity, activity variations with quantity of substrate, temperature, pH, etc. Divalent cations stimulated strongly the ATPase in the following order: Mn²⁺ > Mg²⁺ > Ca²⁺. The maximum ATP hydrolysis velocity for that part of ATPase activity which is strictly dependent on Mg²⁺ ions was 3.85 μmol × mg^?1 protein × h^?1. This plasma membrane ATPase was not sensitive to ouabaïn or to oligomycin.     

The effects of cations and diamines on the viscosity of T2 DNA

The viscosity of the DNA of T2 bacteriophage has been studied with the aim of understanding the folding of the DNA in the phage head. Thus the results of previous workers have been extended into a range of concentrations and mixtures of cations and polyamines simulating the conditions obtaining in actual phage heads. Difficulties in obtaining reproducible results with DNA in 0.001M NaCl have been attributed to the presence of traces of protein, perhaps nulease(s), that can be eliminated by hot-phenol extraction of the DNA. The results have been negative in the sense that the minimum viscosity obtained (120–140 dl/g) indicates a molecule that is far from tightly folded and in that viscosities in this range can be obtained with a simple mixture of Na⁺ and Mg⁺⁺. No specificity of the diamines putrescine and spermidine is seen; they are simply surrogate cations.     

Thermal denaturation of Na- and Li-DNA in salt-free solutions

Thermal denaturation of Na- and Li-DNA from chicken erythrocytes was studied by means of scanning microcalorimetry in salt-free solutions at DNA concentrations (C_p) from 4.5 · 10^?2 to 1 · 10^?3 moles of nucleotides/liter (M). Linear dependencies of DNA melting temperature (T_m) vs lgC_p were obtained: ((1)) ((2)) for Na- and Li-DNA, respectively. Microcalorimetry data were compared with the results of spectrophotometric studies at 260 nm of DNA thermal denaturation in Me-DNA + MeCl solutions at C_p ? (6–8) · 10^?5 M and C_s = 0–40 mM (Me is Na or Li, C_s is salt concentration). It was found that Eqs. (1) and (2) are valid in DNA salt-free solutions over the C_p range 6 · 10^?5?4.5 · 10^?2M. Protonation of DNA bases due to the absorption of CO₂ from air in Na-DNA + NaCl solutions affects DNA melting parameters at C_s < 4 mM. Linear dependence of T_m on lga₊ is found in Na-DNA + NaCl at C_s > 0.4 mMin the absence of contact of solutions with CO₂ from air (a₊ is cation activity). A dependence of [dT_m/dlga₊] on Li⁺ activity was observed in Li-DNA + LiCl solutions at C_s < 10 mM: [dT_m/dlga₊] increases from 17°–18° at C_s > 10 mM to 28°–30° at C_s ? 0.2–0.4 mM. Spectrophotometric measurements at 282 nm show that this effect was caused by protonation of bases in fragments of denatured DNA in neutral solutions. The Poisson–Boltzmann (PB) equation was solved for salt-free DNA at the melting point. The linear dependence of T_m vs lgC_p was interpreted in terms of Manning's condensation theory. PB and Manning's theories fit the experimental data if charge density parameter (ξ) of denatured DNA is in the range 1.8–2.1 (assuming for native DNA ξ = 4.2). Specificity of Li ions in interactions with DNA is discussed. © 1994 John Wiley & Sons, Inc.     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Intrinsic viscosities of cyclic and linear lamda DNA

The ratio of the intrinsic viscosities of the linear and circular forms of λ DNA, [η]L /[η]c, has been measured as a function of ionic strength in the range [Na⁺] = 0.6. M–0.03MCorrections were made for the presence of uncyclizable linear contaminant in circular preparations. By combining data in the literature on the ionic strength dependence of linear DNA of various molecular weights with that obtained here, it was possible to determine the expansion parameter εL as a function of [Na⁺]. εL is defined by the relation 〈L²〉 = b²N^1+εL, where 〈L¹〉 is the mean-square end-to-end distance of a chain of N segments of length b. The empirical relation εL = 0.05 ? 0.11 log [Na⁺] for native NaDNA at 25°C is found. When εL = 0, [η]L /[η]c extrapolates to 1.6, in good agreement with the theoretical prediction of 1.55. As εL increases, [η]L /[η]c increases, in agreement with a theory of Bloomfield and Zimm.     

The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.