The B goes to Z transition of poly(dG-dC) . poly(dG-dC) modified by some platinum derivatives.

Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.     

Circular dichroism of poly(dG-dC) modified by the carcinogens N-methyl-4-aminoazobenzene or 4-aminobiphenyl.

Poly(dG-dC) was modified to different extents by the carcinogens 4-aminobiphenyl (ABP) or N-methyl-4-aminoazobenzene (MAB). HPLC analysis of the enzymatically hydrolyzed modified polymers indicates that more than 90% of the ABP and 81% of the MAB modification occurs at the C8 position of guanine. The conformational changes of the unmodified and modified polymers were studied as a function of ethanol and magnesium ion concentrations by the use of circular dichroism (CD). The modified polymers show a CD inversion pattern similar to that of the salt-induced B to Z transition of poly(dG-dC). Both of the modified polymers require less salt or ethanol than the unmodified polymer for the inversion of the spectra. The amount of ethanol or magnesium needed to induce the inverted CD spectrum is inversely proportional to the percentage of bound ABP or MAB. These data indicate that ABP and MAB can enhance conversion from B to Z conformation in alternating purine-pyrimidine sequences.     

Conformations of poly(dG-dC).poly(dG-dC) modified by the O-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) has been modified by reaction with 4-acetoxyaminoquinoline 1-oxide (Ac-4 HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide. The circular dichroism (CD) spectra of the modified and unmodified polymers have been compared under various experimental conditions. The CD spectra were recorded in 1 mM phosphate, 50% (v/v) ethanol, 3.8 M LiCl and 95% (v/v) ethanol, conditions in which poly(dG-dC).poly(dG-dC) adopts the B-, Z-, C- and A-form respectively. In 1 mM phosphate buffer, poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO seems not to contain regions in the Z-form. Z-form induction could be progressively obtained by the addition of ethanol as follows: in the buffer with about 30% ethanol the modified polymer started to adopt the Z structure, while 40% of ethanol in the buffer was necessary for the unmodified polymer. In the 50% ethanol-1 mM phosphate buffer mixture (v/v), poly(dG-dC).poly(dG-dC) was entirely in the Z-form while poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO remained partially in the B-form. Enzymatic digestions with the nuclease S1 which is specific of the single-stranded DNA were carried out in order to support the modified poly(dG-dC).poly(dG-dC) CD study conclusions. The role played by the two major adducts on the conformational characteristics of modified polymer is discussed.     

Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene.

Poly (dG-dC) . poly(dG-dC) was modified by the reaction with N-acetoxy-N-acetyl-2-aminofluorene. The conformations of poly(dG-dC) . poly(dG-dC) and of poly d(G-C)AAF were studied by circular dichroism under various experimental conditions. In 95% ethanol, the two polynucleotides adopt the A-form. In 3.9 M LiCl, the transition B-form-C-form is observed with poly(dG-dC) . poly (dG-dC) but not with poly d(G-C)AAF. In 1 mM phosphate buffer, poly d(G-C)AAF behaves as a mixture of B- and Z-form, the relative percentages depending upon the amounts of modified bases. The percentage of Z-form is decreased by addition of EDTA and is increased by addition of Mg++. Spermine favors the Z-form in modified and unmodified polynucleotides. No defect in the double helix of poly d(G-C)AAF is detected by SI endonuclease.     

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.     

Effect of chromium(III) on poly(dG-dC) conformation

The interaction of chromium(III) with poly(dG-dC) inhibits the B to Z transition and results in the condensation of the polymer at high Cr/nucleotide ratios. At low Cr/nucleotide ratios chromium(III) enhanced the ability of ethanol to induce the B to Z transition of poly(dG-dC). The effects of chromium(III) on the conformation of DNA may be related to the carcinogenicity of chromium compounds.     

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAS: influence of (G+C) content and nucleotide sequence

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.     

Porphyrin induced Z to B conversion of poly(dG-dC)2 in ethanol

The water soluble porphyrins H2TMpyP-2, H2TMpyP-4, and CuTMpyP-4 are found to bind to Z-form poly(dG-dC)2 in 60% ethanol (v/v) and to facilitate the conversion of the polymer to the B form. Metalloporphyrins with axial ligands (MnTMpyP-4, ZnTMpyP-4) interact to some degree with the Z form, but do not lead to extensive conversion to the B form. The conversion of the Z form into the B form was determined by CD titration experiments, which were used to quantitate the fraction of poly(dG-dC)2 present in each conformation. Under all conditions each bound porphyrin molecule converts multiple base pairs from Z to B. The kinetics of porphyrin reactions with Z-poly(dG-dC)2 in 60% ethanol were measured using two different detection techniques. Stopped flow spectrophotometry was used to observe the time-dependent spectral changes associated with the porphyrins during the reaction. Time-dependent changes in the poly(dG-dC)2 conformation were observed directly using CD. The porphyrin absorbance changes under the conditions of these experiments have a much shorter half time (t1/2 approximately 0.1 to 2 sec) than the CD changes (t1/2 approximately 10 sec). Thus it could be determined that a complex with spectral characteristics similar to those of the porphyrin intercalated into B-form poly(dG-dC)2 is produced while the polymer is predominantly in the Z form.     

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.     

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).     

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.     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Facilitation of the conversion from B to Z conformation in poly(dG-dC) modified by anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide

The transition from B to Z conformation has been studied in poly(dG-dC) covalently modified with racemic anti- or syn-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a strong and a weak carcinogen, respectively. Circular dichroism was used to study the kinetics of the transition after a sudden increase of the ionic strength to 2.7 M NaCl. The results show that the rate of the B to Z transition of poly(dG-dC) in high NaCl concentration is considerably enhanced by bound anti-BPDE and diminished by bound syn-BPDE. The results may be interpreted such that at the binding site of anti-BPDE the base stacking is distorted and made looser, which facilitates the B to Z transition. The partly intercalative nature of the syn-BPDE complexes apparently is effective in reducing the rate of the transition. These properties of the two BPDEs may be relevant to explain their different carcinogenic potencies.     

N-2-acetylaminofluorene modification of poly(dG-m5dC)·poly(dG-m5dC) induces the Z-DNA conformation

Poly(dG-m⁵dC)·poly(dG-m⁵dC) was modified by treatment with N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) and its conformation examined by circular dichroism (CD) and susceptibility to S₁ nuclease digestion. A sample with a modification level of 10% shows a CD spectrum characteristic of the Z form and is resistant to digestion by S₁ nuclease. The relative reactivity of several polymers with N-Aco-AAF was shown to follow the order of ease of formation of Z DNA: poly(dG-m⁵dC)·poly(dG-m⁵dC) > poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC). This suggests that AAF reacts more readily with Z DNA than B DNA.     

Alternative conformations of DNA modified by N-2-acetylaminofluorene

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the “base displacement model” with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S, digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S₁ digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC)·poly(dG-dC). Modification of this copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly-(dG-dC)·poly(dG-dC) at high ethanol or salt concentrations. Poly(dG)·poly(dC), which docs not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modification. Differences in conformation were suggested by single-strand specific nuclease S₁ digestion and reactivity with anticytidine antibodies. Highly modified poly(dG-dC)·poly(dG-dC) was almost completely resistant to nuclease S₁ hydrolysis, while, modified DNA and poly(dG)·poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.     

The CD of ligand-DNA systems. I. Poly(dG-dC) B-DNA.

A systematic theoretical study of the CD of double-stranded poly(dG-dC) and its complexes with small molecules is presented. The intrinsic CD of the polymer and the induced CD of a transition belonging to a molecule bound to DNA are calculated using the matrix method. The calculations show considerable differences between pyrimidine-purine and purine-pyrimidine binding sites, and we find that the induced CD of a groove bound molecule is one order of magnitude stronger than that of an intercalated molecule. The results form a sound basis for interpreting the CD of ligand-DNA systems in terms of molecular geometry, interactions, and spectroscopy.     

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.     

Structural forms and transitions for the complex of mercury(II) with poly(dG-dC)

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury(II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N3 or N7 for r less than or equal to 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D2O stabilized the Z structure for poly(dG-dC) complexed with HgCl2.     

Structural Forms and Transitions for the Complex of Mercury(II) with Poly(dG-dC)

Abstract

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury (II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N 3 orN7forr < 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D,0 stabilized the Z structure for poly(dG-dC) complexed with HgCl₂.