Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies

The effects of covalent modification of poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized to probe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly(dGdC).poly(dGdC) and seven base pairs for poly(dGm5dC).poly(dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Facilitation of the conversion from B to Z conformation in poly(dG-dC) modified by anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide

The transition from B to Z conformation has been studied in poly(dG-dC) covalently modified with racemic anti- or syn-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a strong and a weak carcinogen, respectively. Circular dichroism was used to study the kinetics of the transition after a sudden increase of the ionic strength to 2.7 M NaCl. The results show that the rate of the B to Z transition of poly(dG-dC) in high NaCl concentration is considerably enhanced by bound anti-BPDE and diminished by bound syn-BPDE. The results may be interpreted such that at the binding site of anti-BPDE the base stacking is distorted and made looser, which facilitates the B to Z transition. The partly intercalative nature of the syn-BPDE complexes apparently is effective in reducing the rate of the transition. These properties of the two BPDEs may be relevant to explain their different carcinogenic potencies.     

The effect of intercalating drugs on the kinetics of the B to Z transition of poly(dG-dC)

We have measured the ability of the intercalating drugs proflavine, ethidium bromide, actinomycin D, and bismethidiumspermine to inhibit the salt induced transition of poly(dG-dC) from the B to the Z form. While all of the drugs studied slowed the B to Z transition, the effectiveness of the drugs correlates much better with their DNA binding kinetics than their DNA binding constants. In studies where the binding densities of ethidium and actinomycin were varied we have found that high levels of ethidium, more than 1 per 20 base pairs, were required to inhibit the B to Z transition while low levels of actinomycin, less than 1 per 450 base pairs, reduced the transition rate. Studies of the B to Z transition in the presence of both actinomycin and ethidium suggest that the drugs inhibit the transition by different mechanisms. The results are interpreted in terms of a modification of the kinetic model proposed by Pohl and Jovin in which, depending on the DNA binding kinetics of the drug, the drug may inhibit nucleation and/or propagation of the B to Z transition.     

Stabilization of the Z'' form of poly(dGdC):poly(dGdC) in solution by multivalent ions relates to the ZII form in crystals.

Both multivalent ions and 85% ethanol are required to produce the original Z' form of poly(dGdC):poly(dGdC) in solution. When multivalent impurities are removed by dialysis against 0.5 M NaCl and 1 mM EDTA, the circular dichroism retains features of the standard Z form. Addition of Ca+2 nearly reverses this effect. Analysis by singular value decomposition of near-ultraviolet circular dichroism spectra collected during titrations of polynucleotide in 60% ethanol with multivalent ions reveal that, at concentrations below .5 per nucleotide, they stabilize Z'-like forms in a two-state equilibrium with the Z form. Differences among the Z' spectra produced by the different ions suggest that at least three families of Z' structures exist. Furthermore, comparison with crystal data indicates that the Z' form in solution is related to the ZII form in crystals.     

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.     

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.     

Effect of B-Z transition and nucleic acid structure on the conformational dynamics of bound ethidium dimer measured by hydrogen deuterium exchange kinetics.

Ethidium dimer is shown to bind by intercalation, almost equally well, to the B and Z form of poly[(dG-m5dC)].poly[(dG-m5dC)], whereas the ethidium monomer shows a strong preference for the B form. The hydrogen-deuterium (H-D) exchange kinetics of the ethidium dimer bound to the B and Z form of poly [(dG-m5dC)].poly[(dG-m5dC)] could then be compared. The kinetics of the H-D exchange were strikingly slower when the dye was bound to Z DNA as compared to B DNA. The exchange kinetics were also modified when ethidium dimer was bound to tRNA and to a triple stranded structure. It is proposed that a dynamic fluctuation at the level of the nucleic acid could modulate the dynamic fluctuation at the level of the bound ligand.     

Fluorescence-detected circular dichroism of ethidium bound to poly(dG-dC) and poly(dG-m5dC) under B- and Z-form conditions

The equilibrium binding of ethidium to poly(dG-dC) and poly(dG-m5dC) under conditions favoring B and Z forms was investigated with fluorescence-detected circular dichroism (FDCD) and optical titration methods. FDCD spectra indicate a similar geometry for the intercalated ethidium under both B- and Z-form conditions, even at low levels of bound ethidium. The magnitude of the 310-330-nm FDCD band as a function of the bound drug to base pair ratio (r) indicates ethidium binds to poly(dG-dC) in 4.4 M NaCl and to poly(dG-m5dC) in 25 mM MgCl2 by clustering. Under these conditions, circular dichroism spectra indicate the polymer is largely Z form. Thus, it appears ethidium clusters into regions it has induced into a right-handed form. For all conditions studied, the FDCD spectra provided no evidence for a left-handed binding site. Under B-form conditions, binding is random.     

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.     

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.     

Structure and stability of Z* DNA

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn++, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA. Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent. Poly(dGdC) preparations with average lengths of 60, 240, 500, and 2000 base pairs all form Z* DNA. Poly(dGm5dC) forms Z* DNA in the presence of Mn++ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn++ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique

Fluorescent dansyl labels were covalently attached to poly (L-lysine) (poly(Lys)) with a degree of polymerization of 300 to 600. The degree of labeling was 0.01 to 0.085 (mol label to mol amino acid residues). From the decay of the anisotropy of fluorescence it was concluded that the labels were highly mobile both in the coiled and helical state. A decrease of fluorescence intensity accompanied the helix-coil transition. Identical pH induced transition curves were measured by circular dichroism and fluorescence. The midpoint of the transition was at pH 10.2. The kinetics of the transition were studied by temperature-jump relaxation using fluorescence detection. A single relaxation phase was observed. The relaxation time tau exhibited a distorted bell shaped dependence on the degree of helicity f with a maximum value tau(max) = 15 micros at f = 0.3 and 20 degrees C. It was independent of polymer concentration and of the degree of labeling. A rate constant of helix propagation kF = 10(7) s(-1) was calculated from tau(max) and published values of the nucleation parameter sigma. The activation energy was 16 kJ mol . The observed rate constant is comparable to that of poly(L-glutamic acid) but two orders of magnitude smaller than that found for polyamino acids with nonionizable side chains.     

Temperature-dependent reversible transition of poly(dCdG) · poly(DcdG) in ethanolic and methanolic solutions

Using CD we investigated the transitions of poly(dCdG) · poly(dGdC) from B-to-Z form and from Z-to-Z′ form. We have found experimental conditions that allow the cooperative transition to occur as a function of temperature in ethanolic solutions. The transition is reversible and can be repeated as often as desired. There is no evidence of strand separation during the cooperative transition as monitored by absorbance. For purposes of calculation, we have assumed a two-state model for the B-to-Z transition, although the data indicate that such a model is too simplistic. The calculations allow the estimation of the change in enthalpy per mole of cooperative unit for the transition as a function of ethanol concentration. The values range from ±140 to ±200 kcal/mol for ethanol concentrations between 10 and 20%. Investigations of the noncooperative Z-to-Z′ transition show that it is a reversible two-state transition. The different forms of poly(dCdG) · poly(dGdC) give no scattering contributions to the CD as shown by fluorescent-detected CD or fluorscat techniques. This indicates that the CD spectra are true spectra, and contain no contributions from differential scattering of the polynucleotide. This is particularly significant in the case of the Z′ form, since it exists at high ethanol concentrations (80%) where condensation of polynucleotides can provide large contributions to the CD spectra. Analogous investigations using methanol show that the two transitions also occur, but the final Z′ form in methanol is qualitatively different from the ethanol form.     

Methylation and restriction endonuclease cleavage of linear Z-DNA in the presence of hexamminecobalt (III) ions.

These studies employed the synthetic linear DNA, poly dGdC, in the B and cobalt hexammine chloride (Co)-induced Z form to determine the effect of conformation on protein-DNA interactions. The rate of the reaction of the restriction endonucleases, Hha I and Cfo I, are reduced with Z DNA as compared to B DNA. The ability of both restriction endonucleases to react with an aggregate form of Z DNA (Z* DNA) is found to depend upon how the Z* DNA is formed. When Z* DNA is induced by low concentrations of Co (50 microM), the endonucleases remain active. In the presence of 100 microM Co, which causes increased aggregation, the endonucleases are inactive. The Hha I DNA methyltransferase reacts at equal rates with the B, Z and low cobalt Z* forms and at a greatly reduced rate with the high cobalt Z* form. These results are significantly different than those observed with Z form dGdC tracts inserted into circular DNA molecules.     

Structure and Stability of Z* DNA

Abstract

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn⁺⁺, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent Poly(dGdC) preparations with average lengths of 60,240,500, and 2000 base pairs all form Z* DNA Poly(dGm⁵dC) forms Z* DNA in the presence of Mn⁺⁺ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn⁺⁺ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

The transitions between left- and right-handed forms of poly(dG-dC).

The circular dichroism study of water/trifluoroethanol (TFE) solutions of poly(dG-dC) has revealed the following: The polynucleotide is present as a B form up to a TFE content of 60% (v/v) or less. Then, a cooperative transition into a left-handed Z form occurs. Within the region of 66-78% TFE, a continuous non-cooperative change is going on which can be attributed to an intrafamily transition within the family of Z forms. At last, in the interval of 80-84% TFE, a second cooperative transition, probably, Z - A is realized. Both transitions, Z - A and Z - B, show slow kinetics (10-60 min) while the direct transitions from the A to B form taking less than 10 sec. The length of cooperativity for the B - Z transition, Vo = 25 base pairs was estimated using spermine molecules. Spermine was found to induce the B to Z transition in the (dG-dC) sequences even in the absence of TFE which might be biologically interesting.     

Porphyrin induced Z to B conversion of poly(dG-dC)2 in ethanol

The water soluble porphyrins H2TMpyP-2, H2TMpyP-4, and CuTMpyP-4 are found to bind to Z-form poly(dG-dC)2 in 60% ethanol (v/v) and to facilitate the conversion of the polymer to the B form. Metalloporphyrins with axial ligands (MnTMpyP-4, ZnTMpyP-4) interact to some degree with the Z form, but do not lead to extensive conversion to the B form. The conversion of the Z form into the B form was determined by CD titration experiments, which were used to quantitate the fraction of poly(dG-dC)2 present in each conformation. Under all conditions each bound porphyrin molecule converts multiple base pairs from Z to B. The kinetics of porphyrin reactions with Z-poly(dG-dC)2 in 60% ethanol were measured using two different detection techniques. Stopped flow spectrophotometry was used to observe the time-dependent spectral changes associated with the porphyrins during the reaction. Time-dependent changes in the poly(dG-dC)2 conformation were observed directly using CD. The porphyrin absorbance changes under the conditions of these experiments have a much shorter half time (t1/2 approximately 0.1 to 2 sec) than the CD changes (t1/2 approximately 10 sec). Thus it could be determined that a complex with spectral characteristics similar to those of the porphyrin intercalated into B-form poly(dG-dC)2 is produced while the polymer is predominantly in the Z form.     

Elsamicin A can convert the Z-form of poly[d(G-C)] and poly[(G-m5C)] back to B-form DNA.

The interaction of poly[(G-C)] and poly[d(G-m5C)] with the antitumor antibiotic elsamicin A, which binds to alternating guanine + cytosine tracts in DNA, has been studied under the B and Z conformations. Both the rate and the extent of the B-to-Z transition are diminished by the antibiotic, as inferred by spectroscopic methods under ionic conditions that otherwise favor the left-handed conformation of the polynucleotides. Moreover, elsamicin converts the Z-form DNA back to the B-form. The circular dichroism data indicate that elsamicin binds to poly[d(G-C)] and poly[d(G-m5C)] to form a right-handed bound elsamicin region(s). The transition can be followed by changes of the molar ellipticity at 250 nm, thus providing a convenient wavelength to monitor the Z-to-B conformational change of the polymers as elsamicin is added. The elsamicin A effect might be explained by a model in which the antibiotic binds preferently to a B-form DNA, playing a role as an allosteric effector on the equilibrium between the B and Z conformations, thus favoring the right-handed one.     

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.