Slow exchanging protons in the Z-form of G-C and A-C alternating polymers by using a rapid dialysis method

Using a dialysis method we have measured the hydrogen exchange (HX) kinetics in poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC), poly(dG-br5dC).poly(dG-br5dC) and platinated poly(dA-br5dC).poly(dG-dT) under experimental conditions in which these polymers adopt the Z-conformation. The latter polymer has one slow exchanging proton with a half-time of about 2 h, whereas the other G-C alternating polymers display a slow class of two protons with exchange half-time of about 6 h. These exchange half-times are independent of ionic strength and of the nature of the salt for all these polymers in the Z-form. The slow proton exchange appears to be strongly correlated to the Z-conformation but rather independent of the Z-DNA sequence. The comparison of the proton exchange rates with the corresponding B in equilibrium Z transition rates is not in favour of the same rate limiting step for both processes.     

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.     

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.     

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.     

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.     

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.     

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.     

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)     

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.     

B----Z DNA conformational changes induced by a family of dinuclear bis(platinum) complexes.

The reactions of bis(platinum) complexes of general formula [(PtClm(NH3)3-m)2(NH2(CH2)nNH2)]2(2-m)+ were studied with poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC) and poly(dG).poly(dC). When m = 0 (Complexes II, n = 2,4) the complexes are saturated 4+ cations capable only of electrostatic interactions with the polynucleotide. Where m = 1 the complexes contain two monodentate platinum coordination spheres with the chloride trans to the diamine bridge (Complexes I, n = 2,4, 1,1/t,t). Complexes I give CD spectra characteristic of a 'Z-like' conformation upon reaction with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) but not poly(dG).poly(dC). The B----Z transition appears independent of interplatinum diamine chain length. As little as 1 bis(platinum) complex per 25-30 base pairs is sufficient to observe the Z-like spectrum. Covalent binding is however not a prerequisite for Z-DNA formation because the polyvalent cations II are also very effective in inducing the B----Z transition in either poly(dG-dC).poly(dG-dC) or poly (dG-m5dC).poly(dG-m5dC). In these cases, the concentrations of II required are significantly lower than analogous monomeric agents such as [Co(NH3)6]3+. The possible biological consequences of the Z-DNA induction by bis(platinum) complexes are discussed.     

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.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

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.     

Binding of recA protein to Z-form DNA studied with circular and linear dichroism spectroscopy

Binding of RecA to poly(dG-m5dC) and poly(dG-dC) under B- and Z-form conditions was studied using circular dichroism (CD) and linear dichroism (LD). LD revealed a quantitative binding of RecA to Mg2+-induced Z-form poly(dG-m5dC) with a stoichiometry of 3.1 base pairs/RecA monomer, which is slightly larger than the 2.7 base pairs observed for the B-form. The LD spectra indicate a preferentially perpendicular orientation of DNA bases and a rather parallel orientation of the tryptophan residues relative to the fiber axis in both complexes. The association rate of RecA to Z-form DNA was found to be slower than to B-form. CD measurements showed that the polynucleotide conformation is retained upon RecA binding, and CD and LD confirm that RecA binds to both forms of DNA. The Mg2+-induced Z-form is shown to be retransformed into B-form, both in free and in RecA-complexed polynucleotides by addition of NaCl, whereas the B----Z transition cannot be induced by addition of Mg2+ when the polynucleotide is complexed with RecA. From this it is inferred that RecA does not stabilize the Z-conformation of the polynucleotide but that it can kinetically "freeze" the polynucleotide in its B-conformation. On all essential points, the same conclusions were also reached in a corresponding study of unmethylated poly(dG-dC) with the Z-form induced by Mn2+.     

The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA

The repair of O6-methylguanine present in N-methylnitrosourea (MNU)-treated alternating polynucleotides MNU-poly(dG-dC) X poly(dG-dC) and MNU-poly(dG-me5dC) X poly(dG-me5dC] was investigated using O6-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me5dC) X (dG-me5dC), can adopt the Z-conformation under physiological conditions. The conformational transition of the poly(dG-me5dC) X poly(dG-me5dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The O6-methylguanine residues are repaired in MNU-poly(dG-me5dC) X poly(dG-me5dC) in B-form. At variance, the conversion of this template to the Z-form completely inhibits the repair of the O6-methylguanine residues. The cooperative transition from the Z- to the B-form of MNU-poly(dG-me5dC) X poly(dG-me5dC), mediated by intercalating drugs such as ethidium bromide, restores the ability of MNU-poly(dG-me5dC) X poly(dG-me5dC) to be substrate for the transferase. These results imply that the promutagenic DNA lesion O6-methylguanine persists in Z-DNA fragments and suggest that DNA conformation modulates the extent of DNA repair and, as a result, plays an important role in determining the mutagenic potency of chemical carcinogens.     

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.     

Structural specificity of polyamines in left-handed Z-DNA formation. Immunological and spectroscopic studies

Natural polyamines putrescine, spermidine and spermine are ubiquitous cellular components. Recent studies showed that these compounds are capable of provoking a conformational transition in poly(dG-m5dC).poly(dG-m5dC) from its usual right-handed B-DNA form to a left-handed Z-DNA form at physiologically relevant cationic concentrations. We studied the efficacy of spermidine, six homologs of spermidine (H2N(CH2)nNH(CH2)3NH2, where n = 2 to 8 (n = 4 for spermidine)) and diethylene triamine to provoke the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) using a monoclonal anti-Z-DNA antibody and spectroscopic techniques. The concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 30 +/- 1 microM. Chemical structural effects were significant when the spermidine homologs were used to induce the transition. The midpoint concentration increased as the number of -CH2 groups varied in relation to that of spermidine. We interpret these structural effects on the basis of molecular models of the interaction of polyamines with polynucleotides.     

Molecular aspects on the interaction of aristololactam-beta-D-glucoside with H(L)-form deoxyribonucleic acid structures

Synthetic alternating GC-rich DNA polymers can adopt Hoogsteen base-paired structures (H(L)-form) under the influence of low pH and temperature. The interaction of aristololactam-beta-D-glucoside (ADG), a natural glucoside derivative of aristolochia group of alkaloids, with protonation-induced structures (H(L)-form) of poly(dG-dC).poly(dG-dC) and poly(dG-m(5)dC).poly(dG-m(5)dC) has been studied using different biophysical techniques. The binding of ADG to protonated DNA is characterized by typical hypochromism and bathochromism of the absorption spectrum of the alkaloid, quenching of steady state fluorescence intensity, decrease in quantum yield, increase in fluorescence polarization anisotropy values, increase in thermal transition temperature of polynucleotides following alkaloid binding and perturbation of circular dichroic spectrum of polynucleotides as a result of its interaction with the alkaloid. Scatchard analysis of the data indicates that ADG binds to protonated structures in a nonlinear noncooperative manner. The binding parameters determined from spectrophotometric titration data employing excluded site model indicate that protonated poly(dG-m(5)dC).poly(dG-m(5)dC) is more favorable for ADG binding than the corresponding nonmethylated analog. The binding of ADG to protonated structures renders a higher degree of stabilization against thermal denaturation compared to respective B-form-ADG interactions and induces a conformational switch to a bound altered form which is different from its interaction with B- and Z-form DNA structures. Thermodynamic parameters (Delta G degrees, Delta H degrees and Delta S degrees ) obtained by van't Hoff analysis of the data indicate that the binding of alkaloid to protonated structures is an exothermic process and the binding free energy arises primarily from a negative enthalpy change. Moreover, the binding leads to an increase in the contour length of protonated DNAs. These results suggest that ADG possibly binds to protonated DNAs by the mechanism of intercalation.