Interpretation of 25Mg spin relaxation in Mg-DNA solutions: temperature variation and chemical exchange effects.

The temperature dependencies of line shapes and spin-lattice relaxation times T1 have been measured for 25Mg in dilute solutions of Na-DNA/NaCl containing varying amounts of added magnesium(II) ions. The 25Mg spectrum is clearly non-Lorentzian, due to the presence of motions modulating the quadrupolar interaction that are slow compared to the inverse of the Larmor frequency. The weakly temperature-dependent line shapes and relaxation rates appear to be influenced by the relatively slow exchange of the Mg2+ ions between the DNA surface and the aqueous bulk phase. The observed temperature dependencies depend on the ratio of total magnesium to DNA phosphate, Mg/P. The line shape as well as the temperature dependence of the line width at half height can be qualitatively reproduced with a two-site discrete exchange model for the quadrupolar relaxation of a spin 5/2 nucleus in isotropic solution. The calculations give a value of the lifetime for magnesium bound to DNA of 4 ms at room temperature. Previously reported temperature-dependent 43Ca relaxation measurements in DNA solution can be reproduced under the assumption of a mean lifetime of bound calcium that is not larger than 2 ms but not smaller than 50 microseconds at room temperature. The temperature variation of T1 for 25Mg has been calculated, giving some qualitative agreement with the data. The correlation time for bound 25Mg has been found to be about 40 ns at room temperature.     

25Mg-nmr investigations of the magnesium ion–DNA interaction

²⁵Mg-nmr data are reported that address the nature of the magnesium ion–DNA interaction. It is found that competitor ions such as calcium, mercury, zinc, and cobalt ions are not effective in competing for all of the magnesium ion–DNA interaction that is reported by the ²⁵Mg-nmr spectrum. The temperature dependence of the ²⁵Mg-nmr spectrum in DNA solution studied at high concentrations of competitor ion indicates that the chemical-exchange lifetime of the magnesium ions at DNA binding sites makes a major contribution to the ²⁵Mg-nmr line width. However, the activation parameters are not consistent with the temperature dependence of either transport properties or chemical exchange with phosphate groups alone, but are consistent with a sum of at least two processes that provide opposing contributions to the ²⁵Mg-nmr relaxation. It is also shown that the non-Lorentzian character of the ²⁵Mg-nmr line previously reported is consistent with the effect of an incompletely averaged static nuclear electric quadrupole interaction and/or an exchange process that is slow with respect to the magnitude of this interaction. Because the concentrations employed in these experiments are high, the present data do not provide a direct or critical test of the electrostatic theories of ion–polyelectrolyte interaction. The present data do demonstrate, however, that such theories alone are insufficient as a basis for understanding the ²⁵Mg-nmr data.     

Theory of H1-mediated control of higher orders of structure in chromatin

It is known that the lysine-rich histone H1 induces both higher orders of folding in chromatin and donut shapes in DNA. However, these phenomena occur only on the high-salt side of a narrow transition range located at about 0.02M salt. Previous theoretical analyses of the ionic-strength dependencies of DNA persistence length and denaturation rate have provided the information that the basic rigid-rod unit in high-molecular-weight DNA is a segment 60 base pairs in length and that if the phosphate charge is neutralized, this segment will spontaneously adopt a bent conformation with radius of curvature 170 Å. On the assumption that an H1 molecule does not completely neutralize the DNA charge in its vicinity, the theory has been extended here to determine the onset of spontaneous bending as a function of salt concentration and extent of phosphate neutralization. A salt transition of the kind observed has been found for the realistic value of 82% charge neutralization, with the actual value likely to be in the neighborhood of 90%, as suggested by the measurements of Wilson and Bloomfield.¹ It is recalled that the spacer DNA length in chromatin is of about the same length as the DNA rigid-rod unit. If binding of H1 to the spacer induces, as predicted, a bent conformation of radius about 170 Å, then the observed value of about 150 Å for the outer radius of the solenoid presently thought to be the basic mode of folding for a nucleosome chain can be understood as a reflection of the inherent maximum curvature of DNA in aqueous salt solution.     

DNA orientation in shear flow

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ^?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s^?1, the orientation can be adequately described by the empirical relation: LD^r= –(k₁-G)/(k₂ + G), where k₁is a linear function of the square root of the ionic strength and k₂ depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.     

31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

Chromatin structure studied by linear dichroism at different salt concentrations

We have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl₂. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na⁺, this can be explained as a screening effect due to the bulk concentration of Na⁺. With Mg²⁺ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.     

Assessment of protein rotational diffusion by 13C off-resonance rotating frame spin–lattice relaxation: Effect of backbone and side-chain internal motion

The ¹³C off-resonance rotating frame spin–lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in a variety of experimental situations. The original formalism of James and co-workers (1978) (J. Am. Chem. Soc. Vol. 100, pp. 3590–3594) incorporated random isotropic reorientational motion of a rigid spherical rotor with no provision for backbone or side-chain carbonyl group internal motion. Here we demonstrate that the failure to include such internal motion may lead to erroneous rotational correlation time determinations for overall reorientational motion. The effect becomes severe for protein molecular masses in excess of 100 kD. Inclusion of both backbone and side-chain carbonyl carbon internal motion, using reasonable parameters derived from the literature [R. Levy and M. Karplus (1979), Chemical Physics Letters, Vol. 65, pp. 4–11; G. Careri, P. Fasella, and E. Gratton (1975), Critical Reviews in Biochemistry, Vol. 3, pp. 141–164; G. Lipari, A. Szabo, and R. Levy (1982), Nature, Vol. 300, pp. 197–198], plus corrections for anisotropic tumbling [C. F. Morgan, T. Schleich, G. H. Caines, and D. Michael (1990), Biopolymers, Vol. 29, pp. 469–480] and microscopic viscosity [S. H. Koenig (1980), ACS Symposium. Series, Vol. 127, pp. 157–176], leads to reliable values for the correlation time describing overall protein reorientation up to molecular masses of approximately 1000 kD. © 1993 John Wiley & Sons, Inc.     

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.     

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.     

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.     

The effect of hyperoxia on superoxide production by lung submitochondrial particles

Terbium ion (Tb³⁺), like other rare earth lanthanides, has traditionally been viewed as binding nucleic acids at or near their ionized phosphate groups only. Here evidence is presented from ¹H NMR studies that confirms this mode of binding in Tb³⁺-mono-nucleotide complexes. However, in polynucleotides, we find that Tb³⁺ coordinately binds at two distinct sites, the phosphate moiety and electron donor groups on purine and pyrimidine bases. This two-site binding is best illustrated by complexes of Tb³⁺-polyuridylic acid, where the relative sensitivities of the uracil protons H5 and H6 to induced chemical shift and nuclear spin relaxation are the inverse of that seen in Tb³⁺-uridine monophosphate complexes. These data substantiate recently reported results derived from ultraviolet absorption and fluorescence spectroscopy (D. S. Gross and H. Simpkins, 1981, J. Biol. Chem.256, 9593–9598) that two-site binding is characteristic of the terbium(III)-polynucleotide interaction.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

In vitro cytotoxicity studies of parent and nanoencapsulated Holmium-2,9-dimethyl-1,10-phenanthroline complex toward fish-salmon DNA-binding properties and antibacterial activity

Abstract

In this study, the interaction of Holmium (Ho) complex including 2, 9-dimethyl-1,10-phenanthroline, also called Neocuproine (Neo), [Ho(Neo)₂Cl₃.H₂O], as fluorescence probe with fish-salmon DNA (FS-DNA) is studied during experimental investigations. Multi-spectroscopic methods are utilized to determine the affinity binding constants (K_b) of complex–FS-DNA. It is found that fluorescence of Ho complex is strongly quenched by the FS-DNA through a static quenching procedure. Under optimal conditions in Tris(trishydroxymethyl-aminomethane)–HCl buffer at 25?°C with pH?≈?7.2, intrinsic binding constant K_b of Ho complex is 6.12?±?0.04?×?10⁵ M^?1. Also, the binding site number and Stern–Volmer quenching constant are calculated. There are different approaches, including iodide quenching assay, salt effect and thermodynamical assessment to determine the features of the binding mode between Ho complex and FS-DNA. Also, the parent and starch and lipid nanoencapsulated Ho complex, as potent antitumor candidates, were synthesized. The main structure of Ho complex is maintained after encapsulation using starch and lipid nanoparticles. 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to assess the anticancer properties of Ho complex and its encapsulated forms on human cancer cell lines of human lung carcinoma cell line and breast cancer cell line. In conclusion, these compounds could be considered as new antitumor candidates.

Communicated by Ramaswamy H. Sarma     

Association of short DNA fragments: Steady state fluorescence polarization study

We have studied aggregation/association of monodisperse DNA fragments (ranging from 30–90 base pairs) by steady-state fluorescence polarization of intercalculated ethidium. The method of excitation at different wavelengths in the ethidium absorption spectrum provides information about anisotropic twisting and tumbling mobility of the fragments. We find that end-over-end tumbling rather than axial spinning and internal twisting motions are affected by aggregation/association. The critical concentration for observing the effects of intermolecular interactions is approximately 5 mg DNA/mL at room temperature, independent of fragment length. Association is favored by low temperature and high (> 10 mM) concentration of Mg²⁺. From temperature-and salt-dependence experiments we infer that the “aggregates” are similar to those observed in a recently discovered DNA sol–gel transition [M. G. Fried and V. A. Bloomfield (1984) Biopolymers 23 , 2141–2155]. We also discuss possible arrangements of the fragments within the aggregates and their possible relation to formation of DNA liquid crystals.     

Sampling of Protein Dynamics in Nanosecond Time Scale by 15N NMR Relaxation and Self-Diffusion Measurements

Abstract

This paper presents a procedure for detection of intermediate nanosecond internal dynamics in globular proteins. The procedure uses ¹H-¹⁵N relaxation measurements at several spectrometer frequencies and hydrodynamic calculations based on experimental self-diffusion coefficients. New heteronuclear experiments, using pulse field gradients, are introduced for the measurement of translation diffusion coefficients of ¹⁵N labeled proteins. An advanced interpretation of recently published (Luginbühl et al., Biochemistry, 36, 7305–7312 (1997)) backbone amide ¹⁵N relaxation data, measured at two spectrometers (400 and 750 MHz for ¹H) for N-terminal DNA-binding domain (1–63) of 434 repressor, is presented. Non-applicability of commonly used fast (picosecond) dynamics model (FD) was justified by (i) poor fit of relaxation data by the FD model-free spectral density function both for isotropic and anisotropic models of the overall molecular tumbling; (ii) specific dependence of the overall rotation correlation times calculated from T₁/T₂ ratio on the spectrometer frequency; (iii) mismatch of the ratio of longitudinal ¹⁵N relaxation times T₁, measured at different spectrometer frequencies, in comparison with that anticipated for the IT) model; (iv) significantly underestimated overall rotation correlation time provided by the FD model (5.50±0.15 and 5.80±0.15 ns for 750 and 400 MHz spectrometer frequency respectively) in comparison with correlation time obtained from hydrodynamics. On the other hand, all relaxation and hydrodynamics data are in good correspondence with the model of intermediate (nanoseconds) dynamics. Overall rotation correlation time of 7.5±0.7 ns was calculated from experimental translation self-diffusion rate using hydrodynamics formalism (Garcia de la Torre, J. and Bloomfield, V.A. Quart. Rev. Biophys., 14, 81–139 (1981)). The statistical analysis of ¹⁵N relaxation data along with the hydrodynamic consideration clearly revealed that most of the residues in 434(1–63) repressor are involved in the nanosecond internal dynamics characterized by the the mean order parameters of 0.59±0.06 and the correlation times of ca. 5 ns.     

OnionTree XML: A Format to Exchange Gene-Related Probabilities

Abstract

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC)?(poly)dG-dC) in low salt solutions exist as a right- handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1, 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2′H2” sugar protons; and in the difference NOE spectra in the H2′H2” region the signatures of the right and left-handed helices become imprinted.     

Five novel palladium(II) complexes of 8-hydroxyquinoline and amino acids with hydrophobic side chains: synthesis,characterization, cytotoxicity,DNA- and BSA-interaction studies

Abstract

The side effects and resistance of metal-based anticancer drugs prompted us to synthesis a novel series of five Pd(II) complexes of the type [Pd(8-QO)(AA)]; where 8-QO?=?anion of 8-hydroxyquinoline and AA?=?anions of amino acids having nonpolar aliphatic side chain such as glycine (–H), alanine (–CH₃), valine (–CH(CH₃)₂), leucine (–CH₂–CH(CH₃)₂) and isoleucine (–CH(CH₃)CH₂–CH₃). The complexes have been characterized with the help of FT-IR, UV–Vis, one and two-dimensional ¹H-NMR, elemental analysis and conductivity measurements. On the basis of these characterization data, a four coordinated square planar geometry for all of these complexes have been proposed. The compounds were screened for their in vitro activities against human cancer cell line, MOLT-4 and their 50% inhibition concentration were ascertained by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Since four out of the five newly synthesized compounds were found to be more active than the standard anticancer drug, cisplatin, their detailed interaction with calf thymus DNA (as a target) and bovine serum albumin (BSA) (as a carrier) were also carried out by utilizing absorption spectra, fluorescence spectra and ethidium bromide displacement studies. In these experiments, several binding and thermodynamic parameters were also calculated. These results suggested that hydrogen binding and van der Waals forces play a major role in the interaction between metal complexes with CT-DNA and BSA.

Communicated by Ramaswamy H. Sarma     

Rat polymerase beta gapped DNA interactions: antagonistic effects of the 5' terminal PO4 - group and magnesium on the enzyme binding to the gapped DNAs with different ssDNA gaps

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO ₄ ⁻ group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg⁺² differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO ₄ ⁻ group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO ₄ ⁻ group increases the stability of internal intermediates with the increasing length of the gap, Mg⁺² decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg⁺² effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme. This work was supported by NIH Grant GM-58565 (to W. B.)     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.