Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

DNA interaction with biologically active divalent metal ions: binding constants calculation

In our previous work we have shown that under the action of Cu²⁺, Mn²⁺ and Ca²⁺ ions DNA is able to transit into a compact state in aqueous solution. In the present work we carried out calculations of binding constants for divalent metal ions interacting with DNA in terms of the macromolecule statistical sum. The formula for calculation of the binding constants and cooperativity parameters was proposed. It was shown that on the “coil state”–“compact (globule) state” transition a single DNA molecule may undergo the first-order phase transition while the transition of the assembly of average DNA chains is of sigmoidal character typical of the cooperative and continuous transition.     

Interaction of DNA with divalent metal ions

The interaction between the native DNA macromolecules and Ca2+, Mn2+, Cu2+ ions in solutions of low ionic strength (10(-3) M Na+) is studied using the methods of differential UV spectroscopy and CD spectroscopy. It is shown that the transition metal ions Mn2+ exercise binding to the nitrogen bases of DNA at concentrations approximately 5 x 10(-6) M and form chelates with guanine of N7-Me(2+)-O6 type. Only at high concentrations in solution (5 x 10(-3) M) do Ca2+ ions interact with the nitrogen bases of native DNA. In the process of binding to Ca2+ and Mn2+ the DNA conformation experiences some changes under which the secondary structure of the biopolymer is within the B-form family. The DNA transition to the new conformation is revealed by its binding to Cu2+ ions.     

Interactions of DNA with divalent metal ions. IV. Competitive studies of Mn2+ binding to AT- and GC-rich DNAs

It has been inferred from previous studies that Mn²⁺ ions bind preferentially to G·C base pairs in DNA, and it has even been suggested that this preference for G·C pairs might be responsible for some of the Mn²⁺ specific effects observed in various biochemical reactions. In this paper we investigate the AT/GC preference of Mn²⁺ by direct competition studies in which AT-rich DNA was dialyzed against GC-rich DNA in the presence of varying amounts of Mn²⁺. Analysis of these results demonstrates that over a wide range of Mn²⁺/DNA(P) molar ratios, Mn²⁺ binds to A·T and to G·C base pairs with virtually identical affinity, although in a somewhat different mode. Both the present and previous nmr, uv, CD, and melting studies are discussed in terms of the different modes of binding of Mn²⁺ to single- and double-stranded DNA.     

Winding of the DNA helix by divalent metal ions.

When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp. An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect. When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers. The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of anions and divalent metal ions with phosphoenolpyruvate carboxykinase.

The catalytic activity of phosphoenolpyruvate carboxykinase in rat liver cytosol is stimulated by incubating with Fe2+, Mn2+, Co2+, and Cd2+. When purified, the enzyme no longer responds to Fe2+, Co2+, or Cd2+ but retains a response to Mn2+. Low concentrations of SO4(2-) in the incubation medium with enzyme and divalent transition metal allow stimulation by Fe2+ and Co2+ and enhance the response to Mn2+. Under identical conditions, orthophosphate with Fe2+ is a potent inhibitor of the enzyme (half-maximal inhibition at 50 muM). A thiol is required in the incubation medium for the effects of Fe2+ plus sulfate or orthophosphate to be expressed. The magnitude of these effects depends on the thiol concentration. Dithiothreitol is more effective than GSH and activation by sulfate plus Fe2+ appears to require the reduced form of dithiothreitol. Sulfate ion is not considered to be the physiological Fe2+-activator of P-enolpyruvate carboxykinase in rat liver cytosol, as this function is fulfilled by a newly discovered liver protein. Knowledge concerning the interaction of Fe2+ and sulfate with the enzyme may be useful in examining their interaction between the enzyme, ferrous ion, and this activator protein.     

A study of the molecular mechanism of DNA interaction with divalent metal ions

The interaction of DNA with divalent metal ions: Ba2+, Mg2+, Mn2+, Ni2+, Cu2+ in solutions at different ionic strengths mu was investigated. The combination of following methods: flow birefringence, viscometry, UV-spectroscopy and circular dichroism made possible to follow the state of the secondary and tertiary structure of the DNA molecule during its interaction with ions. The presence of divalent ions in solution affects the hydrodynamic properties of DNA only at low mu. At high mu the difference in the action of mono- and divalent ions disappears. The persistence length of DNA does not change during the experiment. It is shown that the Mg2+ and Ba2+ ions interact only with phosphate groups of DNA but Mn2+, Ni2+, Cu2+ ions interact also with the nitrogen bases of the macromolecule.     

Characterisation and selectivity of divalent metal ions binding by citrus and sugar-beet pectins

A scale of selectivity for the binding of calcium and some heavy metal ions by citrus and sugar-beet pectins was set up by pH-measurements. The same order of selectivity was found for the two pectins, decreasing as follows: Cu²⁺ Pb²⁺ Zn²⁺ > Cd²⁺ Ni²⁺ ≥ Ca²⁺. Binding isotherms for Ca²⁺, Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ ions have shown a greater binding level when the ionic strength decreased and when the pectin concentration increased in the presence of 0.1 M NaNO₃. By comparing binding isotherms, the same order of selectivity was found as by pH-studies. Scatchard plots and Hill index evaluation showed for all ions and all pectins anticooperative interactions in water. In the presence of 0.1 M NaNO₃, citrus pectins displayed cooperative interactions for all metal ions. In contrast, for sugar-beet pectins, cooperative interactions only occured with Cu²⁺ and Pb²⁺. With Ca²⁺, Ni²⁺ and Zn²⁺ sugar-beet pectins displayed Scatchard plots which could not be distinguished from an anticooperative binding. This difference of behaviour could be related to the presence of acetyl groups decreasing the affinity of Me²⁺ for sugar-beet pectins.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Ferritin. Binding of beryllium and other divalent metal ions

Rat liver homogenates in 0.1 M Tris, pH 7.5, were heated to 80 degrees C, cooled immediately, and centrifuged at 24,000 X g, and 7Be2+ was added to the supernatant. Twenty-five per cent of the radioactivity was bound to a single protein. It was purified to homogeneity and identified to be ferritin as judged by different criteria. These were sucrose density gradient centrifugation, electrophoresis in polyacrylamide gel of the native or sodium dodecyl sulfate-treated protein, reactivity to antibodies, isoelectric focusing, and total amino acid composition. Comparative study of the ability of ferritin or apoferritin to bind Cd2+, Zn2+, Cu2+, and Be2+ was conducted by using a gel equilibrium technique, Centifree micropartition technique, and microcentrifuge desalting technique. Ferritin could be saturated with Cd2+ or Zn2+ or Cu2+ but not with Be2+ even after 800 g atoms of Be2+ were bound. None of the bound Be2+ was dialyzable at 4 degrees C in 0.05 Tris acetate buffer, pH 8.5, but at pH 6.5 over 80% of the bound metal ion was dialyzed after 72 h. By contrast, apoferritin bound similar amounts of all four metal ions, some of which were dialyzable. By spectrophotometric titrations at pH 6.5 of Be2+ with sulfosalicylic acid (SSA), BeKDSSA was calculated to be 5.0 X 10(-6) M and by competition of sulfosalicyclic acid and ferritin for Be2+ the BeKDferritin was calculated to be 6.8 X 10(-6) M.     

Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Interaction of divalent metal ions with four-stranded polyriboinosinic acid

Interaction of Mg2+, Ca2+, Cu2+ ions with the four-stranded poly(I) was studied using differential UV and visible spectroscopies. It was shown that, up to concentrations of approximately 0.1 M, Mg2+ and Ca2+ ions do not bind to heteroatoms of hypoxanthine of the four-stranded poly(I). Cu2+ ions interact with N7 (and/or N1) and O6 (through the water molecule of the hydrate shell of the ion). The latter seems to induce the enolization of hypoxanthine the deprotonation of N1, and, as a result, the transition of the four-stranded helix to single-stranded coils. Single-stranded chains form compact particles with an effective radius of about 100 A.     

Simultaneous stochastic sensing of divalent metal ions

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.     

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.