Divalent cations are not required for the stability of the low-salt Z-DNA conformation in poly(dG-ethyl5dC)

It is demonstrated that poly(dG-ethyl5dC) adopts Z form in low-salt solution like poly(dG-methyl5dC). Its existence is, however, not contingent on the presence of divalent cations in the polynucleotide solution. The Z form is transformed into B form below room temperature. The arising B form cannot be transformed back into Z form by millimolar MgCl2 concentrations. On the contrary, the addition of MgCl2 at room temperature converts the low-salt Z form of poly(dG-ethyl5dC) into B form. It follows from the results that Z form is a stable DNA conformation not only at high but even at low ionic strengths.     

Normal-mode calculation for methylated Z-DNA poly(dG-m5dC).(dG-m5dC)

Normal modes of methylated Z-DNA poly(dG-m⁵dC) · (dG-m⁵dC) are computed by helix-lattice dynamics. Good agreement with Raman spectral data is obtained. We discuss improvements in the formulation of the problem that allow us to greatly reduce the size of the matrix used. This leads to greatly reduced calculation times. The improvements come from using knowledge of the C₂ and time-reversal symmetries.     

Poly(amino2 dA-dT) Isomerizes into the Unusual X-DNA Double Helix at Physiological Conditions Inducing Z-DNA in Poly(dG-methyl5dC)

Abstract

It is demonstrated that a two-state conformational isomerization is induced in the poly(amino²-dA-dT) duplex by submillimolar concentrations of divalent magnesium cations in low-salt aqueous solution. The isomerization is fast and has a low degree of cooperativity. The resulting conformer is the unusual X-DNA double helix originally observed with poly(dA-dT) at very high concentrations of CsF. Interestingly, the X form is induced in poly(amino² dA-dT) under the physiological conditions when poly(dG-methyl⁵dC) assumes Z-DNA. The same conditions of stabilization are presumably connected with the fact, observed in previous phosphorus NMR studies, that Z- and X-DNA have similar polydinucleotide backbone architectures. Results presented in this work permit to specify base pair exocyclic groups responsible for the radically different conformational variability of the synthetic DNA molecules containing alternating purine-pyrimidine sequences of GC or AT base pairs.     

Poly(amino2dA-dT) isomerizes into the unusual X-DNA double helix at physiological conditions inducing Z-DNA in poly (dG-methyl5dC)

It is demonstrated that a two-state conformational isomerization is induced in the poly(amino2-dA-dT) duplex by submillimolar concentrations of divalent magnesium cations in low-salt aqueous solution. The isomerization is fast and has a low degree of cooperativity. The resulting conformer is the unusual X-DNA double helix originally observed with poly(dA-dT) at very high concentrations of CsF. Interestingly, the X form is induced in poly(amino2dA-dT) under the physiological conditions when poly(dG-methyl5dC) assumes Z-DNA. The same conditions of stabilization are presumably connected with the fact, observed in previous phosphorus NMR studies, that Z- and X-DNA have similar polydinucleotide backbone architectures. Results presented in this work permit to specify base pair exocyclic groups responsible for the radically different conformational variability of the synthetic DNA molecules containing alternating purine-pyrimidine sequences of GC or AT base pairs.     

B-Z Cooperativity and Kinetics of Poly(dG-m5dC) are Controlled by an Unfavorable B-Z Interface Energy

Abstract

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m⁵dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl₂, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl, in 50 mM Nad, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 ± 0.3 kcal/mol and is length independent Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 ± 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Conformational lability of poly(dG-m5dC):poly(dG-m5dC).

The remarkable conformational lability of poly(dG-m5dC):poly(dG-m5dC) is demonstrated by the observation of an acid-mediated conformational hysteresis. An acid-mediated Z conformation that exists in solutions containing low sodium concentrations that would normally favor the B conformation is described in this report. This Z conformation is reached by an acid-base titration of a B-poly(dG-m5dC):poly(dG-m5dC) solution which is not far from the B-Z transition midpoint. The resulting Z conformation is thermally very stable, with direct melting into single strands at approximately 100 degrees C. In contrast, the B form DNA, initially in solutions of the same ionic strength but without exposure to acidic pH, exhibits a biphasic melting profile, with conversion into the Z form (with high cooperativity) prior to an eventual denaturation into single strands at around 100 degrees C. Cooling experiments reveal that such biphasic transitions are quite reversible. The transition midpoint for the thermally poised B to Z transformation depends strongly on the NaCl concentration and varies with sample batch. The acid-mediated Z form binds ethidium more weakly than its B counterpart, and the ethidium induced Z to B conversion occurs in a step-wise (non-allosteric) fashion without the requirement of a threshold concentration. The acid-mediated as well as the thermally poised Z conformations are reversed by the addition of EDTA, suggesting the involvement of trace amounts of multivalent metal ions.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reexamination of the reported B----Z DNA transition in nucleosomes reconstituted with poly(dG-m5dC).poly(dG-m5dC)

Polynucleosomes with poly(dG-m5dC).poly(dG-m5dC) have been reconstituted, and well-defined nucleosome core particles from these have been prepared. Upon addition of MgCl2 to the levels used to induce the B to Z transition in this highly methylated DNA, significant changes in the circular dichroism spectrum are observed in solutions of these particles. However, such core particles also exhibit a noticeable instability when compared to chicken erythrocyte core particles under the same conditions. The change in circular dichroism can be entirely accounted for on the assumption that only free nucleotide, released by core particle dissociation, undergoes the B----Z transition. Therefore, no evidence has been found for "Z nucleosomes" in these solutions. In fact, the histone-DNA interaction in the nucelosome seems to partially inhibit the B to Z transition of the DNA. The analysis of our results is consistent with a model in which all of the DNA that remains bound to the histone octamer retains the B form.     

Conduction of Monovalent and Divalent Cations in the Slow Vacuolar Channel

The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.     

Toroidal condensation of Z DNA and identification of an intermediate in the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC)

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts.     

Vacuum UV CD of the low-salt Z-forms of poly(rG-dC).poly(rG-dC), and poly(dG-m5dC).poly(dG-m5dC)

The vacuum CD spectra of poly(rG-dC)·poly(rG-dC) and poly(dG-m⁵dC)·poly(dG-m⁵dC) have been obtained for the low-salt Z-conformations of both polymers. The spectra are very similar to those for the high-salt Z-forms. This behavior is consistent with the suggestion that the low- and high-salt Z-forms are comprised of different proportions of Z_I- and Z_II-conformations.     

Conformation and dynamics of a left-handed Z-DNA hairpin: studies of d(CGCGCGTTTTCGCGCG) in solution

The physical properties of the DNA oligomer d(CGCGCGTTTTCGCGCG) in solvents containing 4 M NaClO4 and 0.1 M NaCl were investigated by proton NMR, optical melting, and circular dichroism spectroscopy. Results of these investigations are as follows: (i) The DNA hexadecamer exists as a unimolecular hairpin in either high or low salt. (ii) In high salt the stem region of the hairpin is in the left-handed Z conformation. (iii) In either high or low salt, the duplex stem of the hairpin is stabilized against melting by approximately 40 degrees C compared to the linear core duplex. The added stability of the hairpin is entropic in origin. (iv) In high salt, as the temperature is elevated, the equilibrium structure of the duplex stem of the hairpin shifts from the Z to the B conformation before melting. (v) In low salt, when the DNA duplex exists in the B conformation, attachment of a T4 single-strand loop to one end only slightly decreases (by 14%) the correlation time of the CH5-CH6 interproton vector. In high salt, when the DNA duplex exists in the Z conformation, the correlation time of the CH5-CH6 interproton vector decreases by 51%. Since these viscosity-corrected correlation times are taken to be indicators of duplex motions on the nanosecond time scale, this result directly suggests a larger amplitude of these motions is present in the duplex stem of the hairpin when it exists in the Z conformation.     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.     

B-Z cooperativity and kinetics of poly(dG-m5dC) are controlled by an unfavorable B-Z interface energy

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m5dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl2, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl2 in 50 mM NaCl, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 +/- 0.3 kcal/mol and is length independent. Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 +/- 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event. Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Threshold Occupancy and Specific Cation Binding Modes in the Hammerhead Ribozyme Active Site are Required for Active Conformation

The relationship between formation of active in-line attack conformations and monovalent (Na⁺) and divalent (Mg²⁺) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg²⁺ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg²⁺, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.