Right- and left-handed helixes of poly[d(A-T)].poly[d(A-T)] investigated by infrared spectroscopy

The secondary structures of double-stranded poly[d(A-T)].poly[d(A-T)] in films have been studied by IR spectroscopy with three different counterions (Na+, Cs+, and Ni2+) and a wide variety of water content conditions (relative humidity between 100 and 47%). In addition to the A-, B-, C-, and D-form spectra, a new IR spectrum has been obtained in the presence of nickel ions. The IR spectra of Ni2+-poly[d(A-T)].poly[d(A-T)] films are analyzed by comparison with previously assigned IR spectra of left-handed poly[d(G-C)].poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)], and it is possible to conclude that they reflect a Z-type structure for poly[d(A-T)].poly[d(A-T)]. The Z conformation has been favored by the high polynucleotide concentration, by the low water content of the films, and by specific interactions of the transition metal ions with the purine bases stabilized in a syn conformation. A structuration of the water hydration molecules around the double-stranded Ni2+-poly[d(A-T)].poly[d(A-T)] is shown by the presence of a strong sharp water band at 1615 cm-1.     

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.     

Z form of poly d(A-C).poly d(G-T) in solution studied by Raman spectroscopy.

Poly d(A-C).poly d(G-T) structures have been studied in solution by Raman spectroscopy, in presence of Na+, Mn2+ and Ni2+ counterions. Increase of the Na+ concentration or addition of Mn2+ ions up to 1M MnCl2 does not modify the B geometry of the polynucleotide. On the contrary, in conditions of low water activity (4M NaCl), the presence of small amounts of nickel ions (65 mM) induces a left-handed geometry of the DNA. The shift of the guanine line located at 682 cm-1 in B form to 622 cm-1 reflects unambiguously the C2'-endo/anti-greater than C3'-endo/syn reorientation of the deoxyribose-purine entities. Moreover modifications in the phosphate backbone lines indicate that the polymer is in a Z conformation. New or displaced lines corresponding to adenosine vibrations are correlated with the left-handed structure. An interaction of the Ni2+ ions specifically with the N7 site of purines, combined with a low water activity is necessary to promote the B-greater than Z transition.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.     

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.     

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.     

Left-handed helical structure of poly[d(A-C)].poly[d(G-T)] studied by infrared spectroscopy

Infrared spectroscopic studies demonstrate the ability of poly[d(A-C)].poly[d(G-T)] to adopt a Z-type conformation. The Z form of the unmodified polynucleotide is induced by Ni2+ counterions and not by Na+. The B----Z equilibrium is shifted at room temperature, in the presence of 1 Ni2+/nucleotide, by an increase in the concentration of poly[d(A-C)].poly[d(G-T)]. The importance of specific binding of Ni2+ ions on the N7 site of purines in the stabilization of the Z form is also discussed.     

Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active.

The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner. Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively. The L form of poly[d(G-C)] formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase. B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems. Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.     

Differential promotion and suppression of Z leads to B transitions in poly[d(G-C)] by histone subclasses,polyamino acids and polyamines.

The right-handed (B) conformation of poly[d(G-C)] in 7.5 mM sodium cacodylate and 25% ethylene glycol can be readily converted to the left-handed (Z) conformation by the addition of 250 microM MnCl2 and this transition can be reversed by chelation of the Mn ions with EDTA or by addition of NaCl. This ability to obtain such reversible transitions in solvent and solute conditions which allow DNA-protein interactions and their assessment by c.d. permitted an analysis of the effect of purified histones, polyamino acids, protamine and polyamines on these transitions. Individual core histones H3, H4, H2a and H2b or protamine stabilised the Mn-induced Z form and prevented the transition to B DNA normally observed after chelation with EDTA or on dialysis to physiological salt concentrations. A similar suppression of Z leads to B transition was also achieved with poly-L-arginine (but not with poly-L-lysine). In contrast, histones H1 and H5 promoted the Z leads to B transition. Polyamines (spermine and spermidine) converted the B form to another right-handed (A) form which transformed to the Z form after the addition of EDTA and this Z form was restored to the B conformation on the addition of NaCl. These results suggest that sequence-dependent variations in the conformation of natural DNA may be modulated by interaction with histones and other basic cellular components and may provide a conformational basis for nucleosome formation and possibly for the control of gene expression.     

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)     

Interaction of transition metal ions with Z form poly d(A-C).poly d(G-T) and poly d(A-T) studied by I.R. spectroscopy.

Interactions between Ni2+, Co2+ and purine bases have been studied by I.R. spectroscopy in the case of double stranded regularly alternating purine-pyrimidine polynucleotides poly d(A-T), poly d(A-C).poly d(G-T) and poly d(G-C). The spectra of polynucleotide films have been recorded in hydration and salt content conditions which correspond to the obtention of the classical right-handed (A,B) and left-handed (Z) helical conformations. Selective deuteration of the 8C site of purines has been obtained and is used to detect interactions between the transition metal ions and the adenine or guanine bases. The spectral region between 1500 and 1250 cm-1 corresponding to base in-plane vibrations and involving also the glycosidic linkage torsion is discussed in detail. The selective interaction between the transition metal ion and the 7N site of the purine base is considered to be partly responsible for the stabilization of the base in a syn conformation, which favours the adoption by the polynucleotide (poly d(G-C), poly d(A-C).poly d(G-T) or poly d(A-T)) of a Z type conformation.     

Reversible helix/coil transitions of left-handed Z-DNA structures. Comparison of the thermodynamic properties of poly(dG).poly(dC), poly[d(G-C)].poly[d(G-C)], and poly(dG-m5dC).poly(dG-m5dC)

In contrast to poly(dG).poly(dC), which remains in the B-DNA conformation under all experimental conditions the polynucleotides with the strictly alternating guanine/cytosine or guanine/5'-methylcytosine sequences can change from the classical right-handed B-DNA structure to the left-handed Z-DNA structure when certain experimental conditions such as ionic strength or solvent composition are fulfilled. Up to now the investigation of the helix/coil transition of left-handed DNA structures was not possible because the transition temperature exceeds 98 degrees C. By applying moderate external pressure to the surface of the aqueous polymer solution in the sample cell the boiling point of the solvent water is shifted up the temperature scale without shifting the transition temperature, so that we can measure the helix/coil transition of the polynucleotides at all experimental conditions applied. It can thus be shown that the Z-DNA/coil transition is cooperative and reversible. The Tm is 125 degrees C for poly(dG-m5dC).poly(dG-m5dC) in 2mM Mg2+, 50mM Na+, pH 7.2 and 115 degrees c for poly[d(G-C)].poly[d(G-C)] in 3.04M Na+. The transition enthalpy per base pair was determined by the help of an adiabatic scanning microcalorimeter.     

Interaction of Z form poly(dG-dC).poly(dG-dC) with divalent metal ions: localization of the binding sites by I.R. spectroscopy.

The secondary structures of poly(dG-dC).poly(dG-dC) in the presence of alcaline , alcaline earth and first row transition metal ions (Na+, Mg2+, Co2+, Ni2+) are investigated by infrared spectroscopy. The conformational transitions are studied as a function of the hydration of the polynucleotide and counter-ion nature and content. The use of selectively deuterated poly(dG-dC).poly(dG-dC) on the 8-carbon of guanines allows to show that a direct interaction occurs between the N7 site of guanines and the transition metal ions Co2+ and Ni2+. In the case of Mg2+, for high ion/nucleotide ratios, the interaction occurs essentially at the level of the phosphate groups. This interaction leads to a modification of the left-handed conformation. Based on the IR spectroscopy results, an explanation is proposed for the different efficiencies of these various ions to induce the B----Z transition.     

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides.

The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.     

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).     

Thermodynamics of left-handed helix formation

The thermodynamics of right- and left-handed helix formation by poly[d(G-C)] X poly[d(G-C)] and by poly-(dG-m5dC) X poly(dG-m5dC) were measured spectrophotometrically and calorimetrically. From the spectrophotometric measurements the thermal stabilities of the alternative helical conformations were evaluated as a function of counterion concentration. From the calorimetric measurements the enthalpies of either right-handed or left-handed helix formation were determined. The corresponding experimental delta H values are -8.6 and -11.2 kcal/mol base pairs for the two conformations in poly[dG-C)] X poly[d(G-C)], and -9.0 and -12.7 kcal/mol base pairs, respectively, for poly(dG-m5dC) X poly(dG-m5dC).