Theory of Helix-Coil Transition on DNA-Ligand Complexes: The Effect to Two Types of Interaction of Ligand on the Parameters of Transition

Abstract

The effect of ligand interacting with native DNA by two types on the parameters of helix-coil transition in homopolymers is considered using the most probable distribution method (Yu.S. Lazurkin et al., Biopolymers 1970). It is shown that at a small relative concentration of ligand the melting enthalpy (ΔH) of DNA may be obtained from the universal formula which contains only values directly known from the experiments. It is shown that the formula for the change of melting temperature and width of melting range depending on the total ligand concentration in solution is converted into the corresponding formulae which are defined for the case when only one type of interaction of ligand and DNA is considered.     

The effect of long-range interactions between adsorbed ligands on the DNA helix-coil transition

The influence of different types of long-range interaction of ligands adsorbed on DNA on the helix-coil transition was theoretically considered. The contact interaction was shown to differ significantly from the long-rang one. It was shown also that even weak dependence of a long-range potential on a degree of helicity resulted in the strong changes of a DNA melting curve. This result allowed to understand the different experimental data on DNA melting in the presence of different substances which reduced AT-and GC-base pairs thermostability difference.     

Nucleoprotein melting. I. Theory of helix-coil transition of DNA in the presence of proteins with cooperative character of interaction under conditions of reversible binding]

Helix-coil transition of DNA with attached extended ligands able to interact with one another during adsorption on DNA (cooperative or uncooperative binding) has been considered. The general formulae describing dependence of polymer melting curve on concentration of attached ligands have been obtained. It has been shown that cooperativity of interaction with DNA stipulates for two phase profile of the melting curve. The results obtained show that proteins which interact with DNA cooperatively may cause two phase helix-coil transition under conditions of reversible binding.     

Enthalpy of helix-coil transition of DNA: dependence on Na+ concentration and GC-content

The enthalpy of helix-coil transition of DNA (delta H) is determined from the experiments on DNA melting with ligands by means of absolutely general formula, which contains only values directly known from the experiment (M.D. Frank-Kamenetskii, and A.T. Karapetian, Mol. Biol. USSR 6, 621 (1972)) with the combination of the "area" method (P.O. Vardevanian, et al., Biophysica 28, 130 (1983)). The experimentally obtained data show that delta H depends on both concentration of Na+ in solution and GC-content of DNA and is of high accuracy.     

Influence of ligands characteristic of selective binding to a certain type of base pairs on DNA helix-coil transition I. Model. Theory

The DNA helix-coil transition in the presence of ligands interacting selectively with a certain type or types of base pairs has been considered. A calculation method for estimation the influence of lignads on the melting process for which the knowledge of DNA primary structure is not required was proposed. It has been shown that the reverse temperature shift caused by ligands bound to a given type of base pairs at given kind of regions (helix or coli) is in direct proportion to the fist derivative with respect to the degree of helicity from ratio beta ji/n, where beta ji--number of nitrogen bases of i-type at the regions of j-kind; N--total number of DNA base pairs. It was assumed earlier that this shift was in direct proportion to beta ji/Nj, where Nj--number of base pairs in DNA regions of j-kind. The specificity of lignads interaction with given kinds of bases alters the manner of the melting process of the heteropolynucleotide in comparison with homopolynucleotide only in the case when the DNA primary structure has a strong influence on the position of helix and coli regions along the DNA chain. Only when this conditions is fulfilled the inversion of thermostability of AT- and GC-pairs may affect the shape of the melting curve.     

Effect of ligands with selective type of binding on the DNA helix-coil transition

The influence of the extended interacted under adsorption ligands with a selective binding on the DNA helix-coil transition has been theoretically studied. It was found that contact interaction between ligands or/and their extent give rise to a marked non-linearity of the GC-content dependence of the melting temperature. This non-linearity causes a few features of the dependence of the melting range width on ligand concentration [delta T(C0)]. Such as a non-monotony of the delta T (C0) increase in the presence of ligands increasing the difference between the thermostabilities of poly(d(A-T)] and poly[d(G-C)] polymers. The degree of a non-linearity defines the character of changes of the form of the differential melting curves in the presence of ligands.     

Magnesium ion effect on the helix-coil transition of DNA

The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10^?6–10^?1M at the ionic strengths of 10^?3M Na⁺. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg⁺⁺ increases the DNA stability, the effect being observed mainly in the concentration range 10^?6–10^?4M. At[Mg⁺⁺]>10^?2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ～10^?5M and then decreases to 7–8°C at the ion content of 10^?3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na⁺] = 10^?3M) and [Mg⁺⁺] ? 10^?5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA–Mg⁺⁺ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.     

Theoretical calculations of the helix–coil transition of DNA in the presence of large,cooperatively binding ligands

Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.     

Nucleoprotein fusion. III. Causes and conditions for development of the multiphase character of the DNA--protein fusion curve

The influence of proteins reversibly and irreversibly bound to DNA on the shape of melting curve has been considered. It is shown that the melting curve becomes biphasic in two cases: (i) cooperative binding of proteins with DNA (II) STRONG DIFFERENCE IN THE BINDING CONSTANTS WITH HELICAL AND COILED REGIONS. Simple formulae permitting to determine which of two causes stipulate for biphasic profile of a given experimental melting curve are obtained. Melting curves of DNA-basic oligopeptides complexes have been investigated. It is shown that the oligopeptides, when their chain length does not exceed 10, are able to migrate along DNA and biphasic shape of the melting curve is stipulated by the cooperative manner of their binding with DNA.     

Peculiarities of DNA-proflavine binding under different concentration ratios

Binding of proflavine to calf thymus DNA is investigated by differential scanning calorimetry and spectrophotometry. It is shown that proflavine interacts with DNA by three binding modes. At high DNA—ligand concentration ratios (P/D), proflavine prefers to intercalate into GC-sites but can also insert into other sites. At low P/D ratios, proflavine interacts with DNA by the external binding mode. The parameters of proflavine-DNA complexation have been calculated from spectrophotometric concentration dependences. Thermodynamic parameters of DNA melting have been calculated from differential scanning calorimetry data.     

Effect of low-molecular amines on DNA conformation and stability of the double helix]

Interaction of low-molecular amines (cystamine, cysteamine, cystaphose, asparagine, beta-alanine) with DNA was studied. The amines change the positive circular dichroism (CD) band of DNA as well as temperature and range width of melting. Effect of amines on DNA depends on ionic strength of the solvent, concentration and structure of the ligand. Monamines cause destabilization of DNA double helix followed by stabilization as ligand concentration increases. At concentrations stabilizing the double helix DNA conformation undergoes transition from the B- to C-form. The results obtained enable to relate the stabilizing effect of low-molecular amines and conformational B leads to C-transition to the non-specific interaction of ligand amino groups with DNA phosphates, and the destabilizing effect of monoamines of low concentrations to their interaction with bases, mainly in the denaturated sites of DNA. It is proposed that a stronger effectiveness of amines as compared to monovalent metals in the conformational shift of DNA towards the C-form is due to the additional effect of disturbance of hydrophobic interactions in DNA double helix.     

Determination of the nucleotide content of DNA blocks by measuring the area under the melting curve

A method for determining the nucleotide content of DNA blocks from areas under the melting curve is suggested. It is shown that this method is more simple and the total melting curve is decomposed not to the Gauss components, but it is considered as as superposition of the melting curves of individual blocks. Application of the method for the determination of calf thymus DNA is discussed.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.     

Model studies of DNA-protein interaction. I. Effect of aminocarboxylic and amide groups of amino acids on DNA thermostability and conformation

To elucidate interactions of amino-carboxylate dipole and amide group of amino acids with DNA, glycine and glutamine, concentration dependences of the melting curves and CD spectra of calf thymus DNA at low ionic strength (10(-4) M) Na-citrate have been studied. A considerable increase of the melting temperature delta t1/2 and a decrease of the temperature interval of melting delta t with the rise of glycine concentration were observed without changes in the CD spectrum. A comparison was made between the influence of dipolar glycine ion and isolated amino and carboxylate ions of ammonium acetate. The data obtained indicated the predominance of electrostatic interaction of glycine with DNA phosphates until the ligand concentration was approximately 0.6 M and, apparently, specific interactions of carboxylate ion with guanine at higher glycine concentrations. Destabilizing effect of glutamine on DNA at a concentration of 5.10(-3) M was observed, whereas at higher concentrations two-phase increase of delta t1/2 was shown. Small changes in DNA CD spectrum under the action of glutamine were registered. The comparison data for glutamine and acrylamide showed that DNA destabilizing effect was due to the amide group. The destabilizing effect of amide group can be explained by its interaction with the bases in single-stranded regions of DNA with the formation of two H-bonds. It is possible that the increase of DNA delta t1/2 is the result of the interaction of phosphates both with aminocarboxylate and amide groups of glutamine.     

DNA sequencing and helix-coil transition. I. Theory of DNA melting

An explicit analytic formula accurately describing the melting of a natural DNA is derived. For phage ?X-174 and virus SV-40, the nucleotide sequences of which are known, the formula fits experimental data for the differential melting curve almost within the experimental accuracy.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

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.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.     

On the application of polyelectrolyte “limiting laws” to the helix-coil transition of DNA. I. Excess univalent cations

A general theory of polyelectrolyte solutions is here used to calculate the differences in Gibbs free energy, enthalpy, and entropy between the coil and helix forms of DNA at any temperature and salt concentration. The salt has univalent cations and is assumed present in excess over the base concentration. The results are restricted to sufficiently dilute solutions. It is shown that the salt concentrations effect is entirely entropic in origin. When applied to the melting temperature, the calculations yield a relation between the enthalpy difference at the melting temperature and the slope of the plot of melting temperature vs. the logarithm of the salt concentration. In accord with observation, both the Gibbs free energy difference at any fixed temperature and the melting temperature are predicted to be linear functions of the log of the salt concentration. However, the theory is not in quantitative agreement with enthalpy data. Data on various colligative and transport properties of both helix and coil forms are reviewed in the text and in Appendix B, and good agreement is found with theory for both forms. No attempt is made to explain why the theory is quantitative for these properties but not for heat measurements. Finally, in Appendix A, an approximate calculation is made of the free energy contributions due to ionic effects not associated with the salt concentration.