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.     

Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase

Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

Z → B transition in poly[d(G-m5C)2] induced by interaction with 4′,6-diamidino-2-phenylindole

The Z form of poly[d(G-m⁵C)₂], in presence of Mg²⁺ ion, is found to be transformed into B form upon interaction with 4′,6-diamidino-2-phenylindole (DAPI). The Z → B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6–7 base pairs. An interaction between DAPI and poly[d(G-m⁵C)₂] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. © 1993 John Wiley & Sons, Inc.     

Long-range allosteric effects on the B to Z equilibrium by daunomycin

Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mn2+ and other transition metals at low concentration induce the right-to-left helical transformation of poly[d(G-C)].

The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.     

Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br5C).d(G-T)]

Poly[d(A-br5C).d(G-T)], a synthetic polynucleotide with a 50% A-T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br5C).d(G-T)] coexist in 4.6 M-NaCl at 45 degrees C. Due to slow exchange, two sets of Tim and Gim resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, Tim = 13.4, 14.1 p.p.m. (parts/million); and Gim = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice (R1) and spin-spin (R2) relaxation rates of the exchangeable thymine (Tim) and guanine (Gim) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br5C).d(G-T)] and the mechanism of the B-Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R1 for both Tim and Gim in the B conformation is dominated by exchange with the solvent, with Tim exchanging more rapidly than Gim. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A-T and G-br5C base-pairs exchange independently of each other. B-form poly[d(A-br5C).d(G-T)] is unusual in that there is an acceleration of the Tim exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 M-NaCl dramatically reduces both the Tim and Gim exchange rates (estimated to be less than 2 s-1 at 70 degrees C). Thus, the G-br5C base-pair and, in particular, the A-T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 degrees C where the B and Z-forms are in equilibrium, we find that the B-Z interconversion rates are less than two per second. In the B conformation at 25 degrees C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 degrees high frequency (less than 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation of DNA in chromatin reconstituted from poly [d(A-T)] and the core histones.

Present results provide direct evidence of the nature of a conformational change in DNA when nucleosomes are formed from core histones and poly [d(A-T)]. First, we have found some features which have characteristic aspects of the A like conformation of DNA. Thus, an increased contribution due to a sugar conformation close to C3'-endo puckering is detected in the Raman spectra. In addition, the circular dichroism (C.D.) spectra of reconstituted chromatin with poly [d(A-T)] exhibits an increases intensity at about 262 nm. A second feature acquired by poly [d(A-T)] in nucleosome formation from core histones is related to the presence of a negative band at about 280 nm in the C.D.spectra. The nature of this change is correlated with a DNA conformation characterized by a decreased number of base pairs per turn (28,29). This indicates that these two features of reconstituted nucleosomes reflect the presence of two types of DNA conformations, which overall form is of the B type (22,36).     

Vacuum UV circular dichroism is diagnostic for the left-handed Z form of poly [d(A-C).d(G-T)] and other polydeoxynucleotides.

Circular dichroism spectra are extended into the vacuum UV to about 178 nm for four polydeoxynucleotides of various sequences capable of assuming the left-handed Z form. It is found that each of these polymers, including those with brominated bases and those with the four different bases, have a characteristic negative feature at short wavelengths when in the Z form. In contrast, the B form only has a positive band between 180 and 200 nm. Furthermore, a blue shift of the short wavelength crossover is diagnostic of the B- to Z-form transition for all polymers studied so far. These results confirm that poly[d(A-C).d(G-T)] can assume the Z form in solution at low concentration.     

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.     

Z form of poly d(A-T).poly d(A-T) in solution studied by CD and UV spectroscopies

Near UV CD spectra, UV absorption spectra and their first derivatives have been recorded on poly d(A-T).poly d(A-T) solutions in presence of high NaCl concentration and various amounts of NiCl2. Comparison of the results presented here with those obtained for poly d(G-C).poly d(G-C) and poly d(A-C).poly d(G-T) in comparable conditions, and the I.R. and Raman data on poly d(A-T).poly d(A-T), allows us to assign the new spectra to the Z conformation of poly d(A-T).poly d(A-T) in solution. The mechanism by which nickel ions induce the B----Z interconversion in the presence of high NaCl concentration is discussed.     

Effect of intercalative binding compared to external binding on Z/B equilibrium of poly d(GMe5C) using fluorescent oxazolopyridocarbazoles as probes

Using the fluorimetric determination of the binding isotherms in combination with circular dichroism, we have investigated the effect of the binding of the intercalating chromophore oxazolopyridocarbazole (OPC) to poly d(GMe5C) on B/Z equilibrium, compared to the effect of the external binder OPC derivative pentyl-2-OPC. The intercalating OPC appears to be very efficient in reversing left-handed poly d(GMe5C) into the right-handed conformation, according to a cooperative mode. For each OPC molecule intercalated into the B form, 7 base pairs were switched from the Z to B conformation. In contrast, the binding of the external binder pentyl-OPC resulted in a limited Z to B transition, involving the switch of 1.4 base pairs from the Z to B conformation. Moreover, OPC appears much more efficient than pentyl-OPC in inhibiting both the extent and kinetics of the salt-induced B/Z transition. At low drug to DNA ratio (D/P = 1/50), a 7-fold and 1.5-fold inhibition of the B/Z transition kinetics occurs in the presence of OPC and pentyl-OPC, respectively. These features are discussed in terms of the difference existing between the entropic contribution in the DNA binding of intercalating agents, compared to external binders.     

Delta V0 of the Na(+)-induced B-Z transition of poly[d(G-C)] is positive

The equilibrium between the right- and left-handed conformations of poly[d(G-C)] in aqueous NaCl shifts towards the right-handed (B) form with increasing pressure. The optical density at 290 and 260 nm was determined at 50 and 180 MPa for solutions in which approximately equal amounts of the two conformations were present at 0.1 MPa (atmospheric pressure). Interpretation of the observed changes in terms of a two-state unimolecular reaction mechanism results in an average molar reaction volume (delta V0) equal to 26 cm3 mol-1 at 22 degrees C; that is, the partial molar volume of B form poly[d(G-C)] is smaller than that of the left-handed (Z) form. Based upon the thermodynamics of ion-pair formation in polar solvents, it is proposed that the positive delta V0 reflects a favorable entropy change for the reaction toward the Z conformation. The larger entropy change of the Z form may derive from the release of water molecules from the hydration spheres of the cation and the poly[d(G-C)] due to the formation of ionic interactions with the Z conformer. The delta V0 of the transition is similar in sign and magnitude to the calculated molar volume change of the interaction of Na+ with H2PO4- in water.     

Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC).

The solution properties of the B and Z forms of poly(dG-dC).poly(dG-dC) have been measured by static and dynamic laser light scattering. The radius of gyration, persistence length, translational and segmental diffusion coefficients, and the Rouse-Zimm parameters have been evaluated. The persistence length of the Z form determined at 3 M NaCl is about 200 nm compared to 84 and 61 nm respectively for the B forms of poly(dG-dC).poly(dG-dC), and calf thymus DNA, both determined at 0.1 M NaCl. The data on persistence length, diffusion coefficients and the Rouse-Zimm parameters indicate a large increase in the chain stiffness of Z DNA compared to the B form. These results are opposite to the ionic strength effects on random sequence native DNAs, for which the flexibility increases with ionic strength and levels off at about 1 M NaCl.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.