The high salt form of poly(dG-dC).poly(dG-dC) is left-handed Z-DNA: Raman spectra of crystals and solutions.

The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support

We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.     

Excision of cytosine hydrates from Z-DNA.

Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E. coli endonuclease III. Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC). Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states.     

Interactions of H1 and H5 histones with polynucleotides of B- and Z-DNA conformations

Interactions of chicken H1 and H5 histones with poly(dA-dT), poly(dG-dC), and the Z-DNA structure brominated poly(dG-dC) were measured by a nitrocellulose filter binding assay and circular dichroism. At low protein:DNA ratios, both H1 and H5 bound more Z-DNA than B-DNA, and binding of Z-DNA was less sensitive to interference by an increase in ionic strength (to 600 mM NaCl). H5 histone bound a higher percentage of all three polynucleotides than did H1 and caused more profound CD spectral changes as well. For spectral studies, histones and DNA were mixed in 2.0 M NaCl and dialyzed stepwise to low ionic strength. Prepared in this way or by direct mixing in 150 mM NaCl, complexes made with right-handed poly(dG-dC) showed a deeply negative psi spectrum (deeper with H5 than with H1). Complexes of histone and Br-poly(dG-dC) showed a reduction in the characteristic Z-DNA spectral features, with H5 again having a greater effect. Complexes of poly(dA-dT) and H5, prepared by mixing them at a protein:DNA ratio of 0.5, displayed a distinctive spectrum that was not achieved with H1 even at higher protein:DNA ratios. It included a new negative band at 287 nm and a large positive band at 255 nm, giving the appearance of an inverted spectrum relative to spectra of various forms of B-DNA. These findings may reflect an ability of the different lysine-rich histones to cause varying conformational changes in the condensation of chromatin in DNA regions of highly biased base sequence.     

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

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.     

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.     

During B-Z Transition There is No Large Scale Breakage of Watson-Crick Base Pairs A Direct Demonstration Using 500 MHz 1H NMR Spectroscopy

Abstract

Monitoring of the Watson-Crick GNH1 proton in poly(dG-dC)-poly(dG-dC) at 500 MHz in 90% H₂0:10% D₂o at 30° C as a function of NaCl concentration (1.5 to 3.6 M), demonstrates that the bases retain Watson-Crick pairing throughout the transition. This observation unequivocally demonstrates that during the B-Z transition there is no large scale and detectable base pair opening and that macroscopically the phenomenon can be described as a direct helix to helix transition. We present frame by frame, an energetically sound stereodynamical trajectory for this transfiguration from right-handed B-DNA to left-handed Z-DNA.     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

Isolation of Z-DNA binding proteins from SV40 minichromosomes: evidence for binding to the viral control region

Proteins dissociated from SV40 minichromosomes by increasing NaCl concentration were tested for their binding to Z-DNA [Br-poly(dG-dC)] and B-DNA [poly (dG-dC)]. Z-DNA binding proteins are largely released in 0.2 M NaCl whereas most B-DNA binding proteins are not released until 0.6 M NaCl. Incubation of SV40 minichromosomes with Z-DNA-Sephadex in low salt solution results in proteins with Z-DNA binding activity (PZ proteins). These proteins bind to negatively supercoiled DNAs containing left-handed Z-DNA but not to relaxed DNAs. They compete with anti-Z-DNA antibodies in binding to negatively supercoiled DNAs. The binding is tighter to negatively supercoiled SV40 DNA than to other plasmids, suggesting sequence-specific Z-DNA binding. PZ proteins binding to negatively supercoiled SV40 DNA interfere with cleavage at the Sph I sites, within the 72 bp repeat sequences of the viral control region, but not with cleavage at the Bgl I site, at the origin of replication. Removal of PZ proteins also exposes the Sph I sites in the SV40 minichromosomes while addition of PZ proteins makes the sites inaccessible.     

Antibody recognition of common epitopes on Z-DNA and native DNA brominated under high salt.

Native calf thymus DNA brominated under high salt (4 M NaCl) adopt Z-/analogous conformation as indicated by UV absorption and difference spectra. The bromination of guanine (44%) was twice as effective as cytosine (22%) and induced thermal instability in the modified polymer. The changes are similar to those observed in Z-form of poly(dG.dC).poly(dG.dC). Antibodies were raised in rabbits against these conformationally altered nucleic acids. Both the polymers were highly immunogenic and produced high titer precipitating antibodies which were completely identical in double immunodiffusion. Immunoaffinity purified IgGs showed binding of common conformational epitopes on Z-DNA and brominated DNA. It is evident that on bromination in high salt, regions of native DNA attain Z-/analogous conformation along its strand.     

Reaction of cis-diamminedichloroplatinum (II) and DNA in B or Z conformation

The nature of the adducts and the conformational changes produced in poly(dG-m5dC).poly(dG-m5dC) by cis-diamminedichloroplatinum(II) (cisPt) have been studied. In the reaction of cisPt and B-DNA, the main adduct is bidentate and arises from an intrastrand cross-link between two guanine residues separated by a cytosine. This was deduced from the study of the compounds by t.l.c. after acid hydrolysis of the polymer. The platinated polymer is not digested by S1 nuclease. The antibodies to Z-DNA bind to the platinated polymer with a smaller affinity than to poly (dG-br5dC).poly(dG-br5dC). The c.d. spectrum differs from that of poly(dG-br5dC).poly(dG-br5dC) or poly(dG-m5dC).poly-(dG-m5dC) in Z conformation. It is concluded that the bidentate adduct induces a conformational change from the B form towards a distorted Z form. In the reaction of cisPt and Z-DNA, a monodentate adduct is formed. This adduct stabilizes the Z conformation as shown by c.d. and binding to the anti-Z-DNA antibodies. At room temperature, the second function of the drug can still react with small ligands such as NH4HCO3. By heating, the second function reacts with a guanine residue. A bidentate adduct is formed as in the reaction of cisPt and B-DNA and it induces a transition from the Z form to the distorted Z form.     

Conformational Transitions of Poly(dl-dC) in Aqueous Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i. e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni²⁺ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA- dT) and poly(dA-dC). poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dl-dC) as in the low-salt form (0.1M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl₂ in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.     

Conformational Peculiarities of Polynucleotides with a Nonrandom Base Sequence According to the 1H→ 3H Exchange Rate in C8H Groups of Purinic Residues

Abstract

We have determined the ¹H→³H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dG- dC) and poly(dA-dC)·poly(dG-dT) as well as homopolynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4–6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25°C. The rate constants for adenine (k_A) and guanine (k_G) of polynucleotides were compared with corresponding constants for E.coli DNA, dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution.

Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT)·poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating “wrinkled” DNA model. The conformations of poly(dG-dC)·poly(dG-dC) and poly(dA-dC)·poly(dG-dT), according to the exchange data obtained, are within the B form. For homopolynucleotides in 0.15 M NaCl, the k_A value for poly(dA)·poly(dT) is nearly the same as k_A for B-DNA, which indicates the similarity of their conformations, whereas the k_G value for poly(dG)·poly(dC) is 1.7-fold lower in comparison with the k_G value in B-DNA. This seems to be connected with the existence of B? A conformation equilibrium for poly(dG)·poly(dC) in solution.

The increase of NaCl concentration to 3 M results in a B→Z transition in the case of poly(dG-dC)·poly(dG-dC) and in the shift of B?A equilibrium towards the A-form in the case of poly(dG)·poly(dC), as is evidenced by alterations of their K_G values. Poly(dA-dT)·poly(dA-dT) in 6 M CsF and poly(dA-dC)·poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the “X-type” CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA)·poly(dT) in 6 M CsF corresponds to the “heteronomous” DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.     

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.     

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.     

Conformational transitions of polynucleotides in the presence of rhodium complexes

We studied the effects of hexammine and tris(ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH3)6(3+) provoked a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the psi-DNA form. In addition, we compared the efficacy of Rh(NH3)6(3+) and Rh(en)3(3+) to provoke the Z-DNA conformation in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC.poly(dG-m5dC). The concentrations of Rh(NH3)6(3+) and Rh(en)3(3+) at the midpoint B-DNA----Z-DNA transition of poly(dG-dC).poly(dG-dC) were 48 +/- 2 and 238 +/- 2 microM, respectively. The psi-DNA form of poly(dG-dC).poly(dG-dC) was stabilized at 500 microM Rh(NH3)6(3+). With poly(dG-m5dC).poly(dg-m5dC), both counterions provoked the Z-DNA form at approximately 5 microM and stabilized the polynucleotide in this form up to 1000 microM concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly(dG-dC).poly(dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.