The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

Salt effects on the denaturation of DNA. V. Preferential interactions of native and denatured calf thymus DNA in Na2SO4 solutions of varying ionic strength.

Precision density measurements were performed at 25°C on Na-DNA-Na₂SO₄ mixtures in the presence of either 0.005 m cacodylic acid buffer (pH 6.8) or in the presence of 0.1 m NaOH (pH 12.3). From measurements executed under equilibrium dialysis conditions, the so-called “density increments” (?ρ/?c₂)_μ⁰ for native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were evaluated as a function of Na₂SO₄ concentration. (?ρ/?c₂)_μ⁰ for native DNA was found to decrease almost linearly with ionic strength I^1/2 of the supporting electrolyte. The density increment for Na-DNA at pH 12.6, on the other hand, increases in more or less linear fashion with I^1/2. (?ρ/?c₂)_μ⁰ for heat-denatured DNA at pH 6.8 is not affected very much by increasing salt strength. From density measurements performed on the Na-DNA–Na₂SO₄ mixtures at fixed concentrations of diffusible components, the partial specific volumes ν ₂° of native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were determined as a function of Na₂SO₄ concentration. All ν ₂° values, irrespective of the secondary structure of the DNA, increase with increasing salt concentration although the increase for heat denatured DNA (pH 6.8) is barely noticeable. ν ₂° of both native and heat-denatured DNA (pH 6.8) extrapolates to a value of 0.50_o ml/g at vanishing salt concentration; ν ₂° of DNA in 0.1 m NaOH, on the other hand, assumes the value 0.2_o ml/g. Distribution coefficients of diffusible components, expressed in terms of preferential water and salt interaction, were evaluated from the (?ρ/?c₂)_μ⁰ data, solvent densities, and partial specific volumes of all solution components. All interaction parameters depend strongly on salt concentration and on the conformation of DNA. From data collected and from information available in the literature it is concluded that Na₂SO₄, for instance, displaces water of hydration from native DNA much more readily with increasing salt concentration than does NaCl. The solvation properties of the denatured forms of Na-DNA are rather complex but appear to be in harmony with whatever information can be gathered from the literature.     

Dielectric constant and ionic strength effects on DNA precipitation.

We have investigated the effect of different zwitterionic compounds on DNA precipitation induced by spermine4+. Glycine, beta-alanine, 4-aminobutyric acid, and 6-aminocaproic acid have shown an increasing capacity to attenuate DNA precipitation. This protection effect has been correlated with the dielectric constant increase of their corresponding solutions. Calculations based on these experimental data and counter-ion condensation theory have confirmed the importance of this parameter for DNA-ion interactions and precipitation mechanisms. We have also observed a resolubilization of DNA in the presence of 6-aminocaproic acid at high spermine4+ concentration and in the presence of glycine at high spermidine3+ concentration. This could be explained by an increase of screening effect with polyamine concentration.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.     

Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II

DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).     

Effect of ionic strength of the medium on the structure and aggregation of histone H1 from the calf thymus

In the region of 50 mM NaCl histone HI has the structure with a great number of binding sites with fluorescent probe 1.8-ANS as compared to the structure formed in solution of 0.6-1.0 M NaCl. These sites, however, have a lower constant of binding with the probe and are characterized by a higher surrounding polarity. At local significant increase of the ionic strength histone HI molecules form stable oligomers having hydrophobic cavities. A conclusion is made about the importance of cationic envelope formed by N- and C-ends around the globular "head", for manifesting effective interactions between several molecules of histone HI.     

Phosphopeptides in highly purified calf thymus DNA

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.     

Two DNA ligase activities from calf thymus

Cell extracts from calf thymus contain two DNA ligase activities, separable by hydroxyapatite chromatography and by gel filtration. Their molecular weights, as estimated from sedimentation coefficients and Stokes radii, are M = 175,000 and M = 85,000, respectively. The two activities both require Mg⁺⁺ and ATP as cofactors, and convert nicked circular DNA molecules to a covalently closed form. The larger of the two ligase activities is more heat-stable than the smaller one, and is also active over a broader pH range.     

Ionic strength dependence of the binding of methylene blue to chromatin and calf thymus DNA.

The binding of the intercalating dye methylene blue (MB) to chromatin and to free DNA has been studied as a function of ionic strength at very low binding ratios (1 MB/400 DNA bases) using absorption spectroscopy. With increasing salt concentration MB is displaced from chromatin to a higher extent than from DNA. The free energy change for MB binding to chromatin is found to be approximately 5 kJ/mole lower than for binding to DNA. This difference can be explained by the reduced number of high affinity binding sites in chromatin due to the presence of histone proteins. The difference in binding energy is virtually independent of the degree of chromatin condensation and also of the valence of counter ions, suggesting that neither the affinity for, nor the number of intercalation sites in the linker DNA is markedly changed upon the salt-induced condensation. The unaffected thermodynamics of the linker binding suggests that factors such as DNA superhelicity and the electrostatic influence from the chromatosomes remain unchanged during chromatin condensation.     

Optimum DNA relaxation reaction conditions for calf thymus DNA-Topoisomerase I are determined by specific enzyme features

Reactivity and chemical properties of calf thymus Topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one. We conclude that:

Experimental and computational studies on the effects of valganciclovir as an antiviral drug on calf thymus DNA

DNA-binding properties of an antiviral drug, valganciclovir (valcyte) was studied by using emission, absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, and computational studies. The drug bound to calf thymus DNA (ct-DNA) in a groove-binding mode. The calculated binding constant of UV-vis, K_a, is comparable to groove-binding drugs. Competitive fluorimetric studies with Hoechst 33258 showed that valcyte could displace the DNA-bound Hoechst 33258. The drug could not displace intercalated methylene blue from DNA double helix. Furthermore, the induced detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity confirm the groove-binding mode. In addition, an integrated molecular docking was employed to further investigate the binding interactions between valcyte and calf thymus DNA.     

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Th?mmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.     

Studies on the interaction of isoxazolcurcumin with calf thymus DNA

The interaction of isoxazolcurcumin (IOC), a synthetic derivative of curcumin, with calf thymus-DNA (ct-DNA) has been investigated by UV-Vis, fluorescence, circular dichroism spectroscopies, viscosity measurements and docking studies. From these analyses, the binding constant, number of binding sites and mode of binding of IOC to ct-DNA has been determined. The binding constant of IOC to DNA calculated from both UV-Vis and CD spectra was found to be in the 10(4)M(-1) range. Analyses of fluorescence spectra, viscosity measurements and molecular modeling of IOC-DNA interactions indicate that IOC is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide displacement studies revealed that IOC did not have any effect on ethidium bromide bound DNA which is indicative of groove binding. To elucidate the preferred region of binding of IOC to DNA, docking studies have been performed and changes in accessible surface area (DeltaASA) of nucleobases determined due to IOC-DNA complexation.     

Interaction of metallopyrazoliumylporphyrins with calf thymus DNA.

The interaction of transition metal complexes of cationic porphyrins bearing five membered rings, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP, M=Mn(III), Ni(II), Cu(II) or Zn(II)), with calf thymus DNA (ctDNA) has been studied. Metalloporphyrins NiPzP and CuPzP are intercalated into the 5'GC3' step of ctDNA. MnPzP is bound edge-on at the 5'TA3' step of the minor groove of ctDNA, while ZnPzP is bound face-on at the 5'TA3' step of the major groove of ctDNA. The binding constants of the metalloporphyrins to ctDNA range from 1.05x10(5) to 2.66x10(6) M(-1) and are comparable to those of other reported cationic porphyrins. The binding process of the metallopyrazoliumylporphyrins to ctDNA is endothermic and entropically driven. These results have revealed that the kind of central metal ions of metalloporphyrins influences the binding characteristics of the porphyrin to DNA.