Structure of the hydration shells of oligo(dA-dT).oligo(dA-dT) and oligo(dA).oligo(dT) tracts in B-type conformation on the basis of Monte Carlo calculations

Monte Carlo simulations [(N, V, T)-ensemble] were performed for the hydration shell of poly(dA-dT).poly(dA-dT) in canonical B form and for the hydration shell of poly(dA).poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero-inclined base pair plane (B' conformation). We introduced helical periodic boundary conditions with a rather small unit cell and a limited number of water molecules to reduce the dimensionality of the configuration space. The coordinates of local maxima of water density and the properties of one- and two-membered water bridges between polar groups of the DNA were obtained. The AT-alternating duplex hydration mirrors the dyad symmetry of polar group distribution. At the dApdT step, a water bridge between the two carbonyl oxygens O2 of thymines is formed as in the central base-pair step of Dickerson's dodecamer. In the major groove, 5-membered water chains along the tetranucleotide pattern d(TATA).d(TATA) are observed. The hydration geometry of poly(dA).poly(dT) in canonical B conformation is distinguished by autonomous primary hydration of the base-pair edges in both grooves. When this polymer adopts a conformation with highly inclined bases and narrow minor groove, the water density distribution in the minor groove is in excellent agreement with Dickerson's spine model. One local maximum per base pair of the first layer is located near the dyad axis between adjacent base pairs, and one local maximum per base pair in the second shell lies near the dyad axis of the base pair itself. The water bridge between the two strands formed within the first layer was observed with high probability. But the water molecules of the second layer do not have a statistically favored orientation necessary for bridging first layer waters. In the major groove, the hydration geometry of the (A.T) base-pair edge resembles the main features of the AT-pair hydration derived from other sequences for the canonical B form. The preference of the B' conformation for oligo(dA).oligo(dT) tracts may express the tendency to common hydration of base-pair edges of successive base pairs in the grooves of B-type DNA. The mean potential energy of hydration of canonical B-DNA was estimated to be -60 to -80 kJ/mole nucleotides in dependence on the (G.C) contents. Because of the small system size, this estimation is preliminary.     

Structure of the hydration envelope of the B-form of polydeoxyribonucleotides poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dTs-dT) from the data of Monte-Carlo simulation

The results of a Monte Carlo simulation of the hydration shell of two polynucleotides poly (dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT) are reported. This study is a part of a series of Monte Carlo computations of the hydration of regular polydeoxyribonucleotides with dinucleotide repeat aimed at looking for dependences of hydration shell structure on base sequence. The coordinates of the main local maximal of water density near the polymers and the topology of the most probable one- and two-membered water bridges are published. For most of the sequences a common primary hydration of base edges of successive base pairs is characteristic. The AT-homopolymeric sequence represents an exception with autonomous primary hydration of a base pair in both grooves, which correlates with the sequence-dependent flexibility and the occurrence of bends of DNA.     

Hoechst 33258--poly(dG-dC).poly(dG-dC) complexes of three types

It was found recently that Hoechst 33258, a dsDNA fluorescent dye used in cytological studies, is an efficient inhibitor of the interaction of TATA-box-binding protein with DNA, DNA topoisomerase I, and DNA helicases. In addition it proved to be a radioprotector. Biological activity of Hoechst 33258 may be associated with dsDNA complexes of not only monomeric, but also dimeric type. In this work, the Hoechst 33258 interaction with poly(dG-dC).poly(dG-dC) was studied using UV-vis and fluorescent spectroscopy, circular and flow-type linear dichroism. It was found that Hoechst 33258 formed with poly(dG-dC).poly(dG-dC) complexes of three types, namely, monomeric, dimeric, and, apparently, tetrameric, and their spectral properties were studied. Complexes of monomeric and dimeric types competed with distamycin A, a minor groove ligand, for binding to poly(dG-dC).poly(dG-dC). We proposed that Hoechst 33258 both monomers and dimers form complexes of the external type with poly(dG-dC).poly(dG-dC) from the side of the minor groove.     

Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms

The enzymatic methylation of chemically alkylated DNA and of poly(dG-dC) X poly(dG-dC) by beef brain DNA(cytosine-5-)-methyltransferase have been tested. The alkylation by dimethylsulfate, which yields mostly 7 methylguanine (m7G) and 3 methyladenine (m3A) do not affect the enzymatic methylation. The dimethylsulfate alkylated poly(dG-dC) X poly(dG-dC) converted into the Z-form in the presence of MgCl2, is just as well methylated as the native or the alkylated polynucleotide in the B-form. The alkylation of DNA or of poly(dG-dC) X poly(dG-dC) by methylnitrosourea yields, in addition to the above base modifications described for dimethylsulfate, methylphosphotriesters and O6-methylguanine. The enzymatic methylation of these substrates modified by methylnitrosourea is decreased. This decrease is proportional to the extent of the chemical alkylation of the substrate.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

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

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

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.     

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.     

Fibre X-ray study of the helix-helix transitions of poly(dG-dC).poly(dG-dC).

Poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) present helix-helix transitions which are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2')-endo and C(3')-endo sugar puckering. Dihedral angles, sets of atomic co-ordinates and stereo views of the two S-DNA structures are given together with curves of calculated diffracted intensities.     

An immunochemical examination of acetylaminofluorene-modified poly(dG-dC) X poly(dG-dC) in the Z-conformation

Immunization of rabbits with a complex of methylated bovine serum albumin and N-2-acetylaminofluorene (AAF)-modified poly(dG-dC) X poly(dG-dC), a polynucleotide that can assume the Z-DNA conformation, yielded several populations of antibodies specific for Z-DNA determinants. The Z-DNA determinants were analyzed by examination of the antisera and of antibody preparations purified on immunoadsorbents. The following was found: AAF-poly(dG-dC) X poly(dG-dC) shared Z-DNA determinants in common with poly(dG-dC) X poly(dG-dC) in 3.0 M NaCl, poly(dG-m5dC) X poly(dG-m5dC) in 1.5 M NaCl, and brominated poly(dG-dC) X poly(dG-dC) in 0.2, 1.5, and 3.0 M NaCl. Included among the antibodies induced by these determinants was a subpopulation whose reaction with brominated poly(dG-dC) X poly(dG-dC) was sensitive to increased ionic strength. Another distinct population of antibodies recognized determinants present on AAF-poly(dG-dC) X poly(dG-dC) but not on the other Z-DNAs. Only a small portion of this population was specific for the AAF moiety; the greater part appeared to recognize Z-DNA-associated conformational characteristics that were unique to AAF-poly(dG-dC) X poly(dG-dC). These findings are consistent with the existence of a continuum of Z-DNA determinants, which might be capable of functioning as recognition signals for regulatory DNA-binding proteins.     

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.     

Dependence of the hydration shell structure in the minor groove of the DNA double helix on the groove width as revealed by Monte Carlo simulation.

The hydration shell of several conformations of the polynucleotides poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) has been simulated using the Monte Carlo method (Metropolis sampling). Calculations have shown that the structure of the hydration shell of the minor groove greatly depends on its width. In conformations with a narrowed minor groove, the first layer of the hydration shell of this groove has only one molecule per nucleotide pair that forms H bonds with purine N3 of one pair and pyrimidine O2 of the next pair. The second layer of the hydration shell of such conformations contains molecules that form H bonds between two adjacent molecules of the first layer. The probability of formation of hydration spine is about 20% while the bridges of the first layer are formed with a probability of about 70%. In the first layer of the minor groove of the B-DNA conformation with wide minor groove there are approximately two water molecules per base pair that form H bonds with purine N3 or pyrimidine O2 and with the sugar ring oxygen of the adjacent nucleotide. The probability of simultaneous H bonding of a water molecule with N3 (or O2) and O of sugar ring is about 30%. The results of simulation suggest that hydration spine proposed for the narrowed minor groove of oligonucleotide crystals [H. R. Drew, and R. E. Dickerson (1981) Journal of Molecular Biology, Vol. 151, pp. 535-556] can be formed in fibers of poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) as well as in DNA fragments of these sequences in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

Spectrophotometric and kinetic studies of the binding of Ni, Co, and Mg to poly(dG-dC) · poly(dG-dC). Determination of the stoichiometry of the Ni-induced B → Z transition

Data are reported for the binding of Ni²⁺, Co²⁺, and Mg²⁺ to the B-form of double-stranded poly(dG-dC) at ionic strength conditions I = 0.001 M, 0.01 M, and 0.1 M. The apparent binding constants for Ni²⁺ and Co²⁺ are about the same and are 2- to 3-fold higher than those for Mg²⁺. Kinetic studies indicate that Mg²⁺ binds to the polynucleotide mainly (or solely) as a mobile cloud (electrostatically, outer-sphere), whereas the transition metal ions undergo site binding (inner-sphere coordination) with poly(dG-dC). The kinetic data suggest that an Ni²⁺ ion coordinates to more than one binding site at the polynucleotide, presumably to G-N₇ and a phosphate group.

At low ionic strength conditions the addition of Ni²⁺ induces a B → Z conformational transition in poly(dG-dC). As demonstrated by UV absorption and CD spectroscopy, the transition occurs at I = 0.001 M already when 3 × 10⁻⁵ – 7 × 10⁻⁵ M of Ni²⁺ are added to 8 × 10⁻⁵ M (in monomeric units) of poly(dG-dC), and at I = 0.01 M between 2.5 × 10⁻⁴ and 4.5 × 10⁻⁴ M of Ni²⁺. Using murexide as an indicator of the concentration of free Ni²⁺ ions, the amount of Ni²⁺ which is bound to the polynucleotide could be determined. At I = 0.001 M it was established that the B → Z transition begins when 1 Ni²⁺ is bound coordinatively per four base pairs, and the transition is complete when 1 Ni²⁺ is bound coordinatively per three base pairs. It is this coordinated Ni²⁺ which induces the B → Z transition.     

Circular dichroism of polynucleotides: Interactions of NiCl2 with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) in a water-in-oil microemulsion

The thermal behavior of the synthetic, high molecular weight, double stranded polynucleotides poly(dA-dT).poly(dA-dT) [polyAT] and poly(dG-dC).poly(dG-dC) [polyGC] solubilized in the aqueous core of the quaternary water-in-oil cationic microemulsion CTAB|n-pentanol|n-hexane|water in the presence of increasing amounts of NiCl(2) at several constant ionic strength values (NaCl) has been studied by means of circular dichroism and electronic absorption spectroscopies. In the microemulsive medium, both polynucleotides show temperature-induced modifications that markedly vary with both Ni(II) concentration and ionic strength. An increase of temperature causes denaturation of the polyAT duplex at low nickel concentrations, while more complex CD spectral modifications are observed at higher nickel concentrations and ionic strengths. By contrast, thermal denaturation is never observed for polyGC. At low Ni(II) concentrations, the increase of temperature induces conformational transitions from B-DNA to Z-DNA form, or, more precisely, to left-handed helical structures. In some cases, at higher nickel concentrations, the CD spectra suggest the presence of Z'-type forms of the polynucleotide.     

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.     

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.     

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.     

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).     

Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.