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.     

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.     

Ethidium binding to left-handed (Z) DNAs results in regions of right-handed DNA at the intercalation site

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.     

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.     

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)     

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.     

Does anthramycin bind to Z-DNA as well as to B-DNA? A molecular mechanics study

Molecular mechanics simulations have been presented on the covalent complexes between anthramycin and the decanucleotides d(GCGCGCGCGC)2 and d(GCGCGTGCGC).d(GCGCACGCGC) in the novel Z form, with the drug docked in the minor groove. The simulations predict that anthramycin binds to Z-DNA almost as well as it does to B-DNA, but with a few key differences in the structural aspects of binding. In particular, the 5' orientation of the drug is preferred in the Z form, contrasting with the preference for 3' orientation in the B form and the drug has a left-handed twist. While no experimental studies have been published on the binding of pyrrolo(1,4)benzodiazepines to left-handed forms of DNA, the energy refined stereochemically satisfactory models provide valuable information which will hopefully simulate high resolution 2-D NMR/NOE studies in solution.     

Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway

recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix. This represents a kinetic rather than an equilibrium effect. Binding to Z DNA is much faster than binding to B DNA. In other respects, binding to the two DNA forms is quite similar. recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs. The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound. There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA. At steady state, the binding favors B DNA in competition experiments. The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

Allosteric conversion of Z DNA to an intercalated right-handed conformation by daunomycin

Absorbance and fluorescence methods were used to measure the binding of the anticancer drug daunomycin to poly (dGdC) under ionic conditions that initially favor the left-handed Z conformation of the polymer. Drug binding was cooperative under these conditions and may be fully accounted for by an allosteric model in which the drug binds preferentially (but not exclusively) to the right-handed B conformation and shifts the polymer from the Z to an intercalated right-handed conformation. Quantitative analysis of binding isotherms in terms of the allosteric model allowed for estimation of the equilibrium constants for the conversion of a base pair at a B-Z interface from the Z to the B conformation and for the formation of a base pair in the B conformation within a stretch of helix in the Z conformation. The free energy of the Z to B conversion of a base pair was calculated from this data and ranges from +0.03 to +0.3 kcal/mol over the NaCl range of 2.4-3.5 M. The free energy for the formation of a B-Z junction was nearly constant at +4.0 kcal/mol over the same range of NaCl concentrations. The salt dependence of the free energy of the Z to B transition indicates preferential Na+ binding to the Z form and that there is a net release of Na+ upon conversion of a base pair from the Z to the B conformation. The energetically unfavorable Z to B transition was found by this analysis to be driven by coupling to the energetically favorable interaction of daunomycin with B form DNA. In 3.5 M NaCl, for example, the free energy change for the overall reaction (Z DNA base pairs) + (daunomycin) in equilibrium with (right-handed complex) is -7.0 kcal/mol, nearly all of which is contributed by the binding of drug to B DNA. Analysis using the allosteric model also shows that the number of base pairs converted from the Z to the B conformation per bound drug molecule is salt dependent and provides evidence that drug molecules partition into regions of the polymer in the right-handed conformation.     

Conversions of the left-handed form and the protonated form of DNA back to the bound right-handed form by sanguinarine and ethidium: a comparative study

The interaction of sanguinarine and ethidium with right-handed (B-form), left-handed (Z-form) and left-handed protonated (designated as H(L)-form) structures of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) was investigated by measuring the circular dichroism and UV absorption spectral analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H(L)-form back to the bound right-handed form. Circular dichroic data also show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation either in Z-form or in H(L)-form. Both the rate and extent of B-form to Z-form transition were decreased by sanguinarine and ethidium under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. The rate of decrease is faster in the case of ethidium as compared to that of sanguinarine. Scatchard analysis of the spectrophotometric data shows that sanguinarine binds strongly to both the polynucleotides in a non-cooperative manner under B-form conditions, in sharp contrast to the highly-cooperative binding under Z-form and H(L)-form conditions. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H(L)-form conditions is associated with a sequential conversion of the polymer from a left-handed to a bound right-handed conformation. Determination of bound alkaloid concentration by spectroscopic titration technique and the measurement of circular dichroic spectra have enabled us to calculate the number of base pairs of Z-form and H(L)-form that adopt a right-handed conformation for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z-form of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) switch to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complexes with H(L)-form of these polynucleotides. Comparative binding analysis shows that ethidium also converts approximately 2 bp of Z-form or H(L)-form to bound right-handed form under same experimental conditions. Since sanguinarine binds preferentially to alternating GC sequences, which are capable of undergoing the B to Z or B to H(L) transition, these effects may be an important part in understanding its extensive biological activities.     

Ternary interactions of spermine with DNA: 4''-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.     

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.     

Evidence for the reversible binding of paraquat to deoxyribonucleic acid

Evidence for the reversible binding of paraquat to calf thymus DNA has been obtained using equilibrium dialysis and thermal melting point determinations. The data indicated the presence of at least two populations of binding site with affinity constants of 6.2 X 10(4) and 7.1 X 10(3) M-1, respectively. The binding capacities of DNA for paraquat were 66 and 480 nmol/mumol DNA nucleotide, respectively, and were equivalent to one ligand bound per 2 DNA phosphate groups. Putrescine inhibited paraquat binding to the low affinity sites without altering binding to the high affinity sites. Scatchard plots of paraquat binding characteristics indicated the presence of positive cooperativity between the compound and DNA. Thermal melting curves of DNA in the presence of paraquat and the endogenous amines putrescine, spermidine and spermine, provided evidence that paraquat cross-linked to DNA with a similar affinity as spermidine. The thermal melting point data also suggested the presence of positive cooperativity between ligand and macromolecule that possibly resulted from a conformation change in the structure of the DNA molecule. Paraquat competitively inhibited the binding of ethidium bromide to DNA and this effect was reversed by Na+. From the data, it is suggested that paraquat binds primarily to the negatively charged phosphates on the DNA backbone but is displaced into the interbase region occupied by the intercalator ethidium bromide. DNA binding of paraquat may, in part, account for its weak mutagenic activity.     

Comparison between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinum (II): immunological and spectroscopic studies

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Inhibition of the thermally driven B to Z transition by intercalating drugs

Poly(dG-m5dC) in phosphate buffer containing 50 mM NaCl and Mg2+ will undergo a reversible thermally driven conversion from the B to the left-handed Z conformation. The temperature at the midpoint of the thermally driven B to Z transition (denoted Tz) is dependent upon the total Mg2+ concentration, with [d(1/Tz)]/(d ln [Mg]) = 0.0134 K-1. The Mg2+ concentration at the midpoint of the equilibrium B to Z transition curve, denoted [Mg]1/2, is dependent on temperature, with (d ln [Mg]1/2)/(d ln T) = -1.02. Binding of the anticancer drug daunomycin to the polymer results in a pronounced increase in Tz, dependent on the molar ratio of added drug. Tz is increased by 71.9 degrees C with nearly saturating amounts of drug bound. Transition profiles are biphasic at less than saturating amounts of bound drug. By experiments monitoring such biphasic curves at a visible wavelength sensitive to the binding of daunomycin, it may be demonstrated that no drug is released until the later phase of the transition. These results are analogous to the effects of intercalating drugs on the thermal denaturation of DNA and indicate that drug molecules preferentially interact with B-form DNA and are redistributed to regions in the B conformation over the course of the transition. Comparative studies show that some intercalators stabilize right-handed DNA more effectively than others. At similar initial binding ratios, the following order, from most to least effective, was experimentally observed: actinomycin greater than daunomycin greater than ethidium greater than proflavin.     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.     

DNA-ethidium reaction kinetics: demonstration of direct ligand transfer between DNA binding sites.

Study of the relaxation kinetics of the interaction of ethidium and DNA reveals a novel and potentially important general binding mechanism, namely direct transfer of the ligand between DNA binding sites without requiring dissociation to free ligand. The measurable relaxation spectrum shows three relaxation times, indicating that three bound dye species are present at equilibrium; about 80% of the dye is in the major intercalated form. For each relaxation the reciprocal relaxation time varies linearly with concentration up to very high DNA concentrations. The failure of the longer relaxation times to plateau at high concentration can be accounted for by including a bimolecular pathway for conversion from one complex form to another. This we envisage as direct transfer of an ethidium molecule, bound to one DNA molecule, to an empty binding site on another DNA molecule. Additional evidence for this direct transfer mechanism was obtained from an experiment showing that DNA (which binds ethidium relatively rapidly) accelerates the binding of ethidium to poly(rA) · poly(rU), presumably by first forming a DNA-ethidium complex and then transferring the ethidium to RNA. The bimolecular rate constant for transfer is found to be about four times larger than the constant for intercalating the free dye. The transfer pathway thus provides a highly efficient means for the ligand to equilibrate over its DNA binding sites, especially at high polymer concentration. The potential importance of direct transfer for DNA-binding regulatory proteins is emphasized.     

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.