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.     

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.     

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.     

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.     

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.     

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)     

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.     

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.     

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 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.     

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).     

Interpretation of DNA vibration modes: I--The guanosine and cytidine residues involved in poly(dG-dC).poly(dG-dC) and d(CG)3.d(CG)3

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5'-dGMP and 2'-deoxycytidine molecules to be reproduced. The role of both C2'-endo and C3'-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (X), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm-1 and 785-500 cm-1 regions, when the right----left-handed conformational transition occurs.     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

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.     

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.     

Comparison of the conformation of poly(dI-dC) with poly(dI-dbr5C) and the B and Z forms of poly(dG-dC). One- and two-dimensional NMR studies

One- and two-dimensional nuclear Overhauser effects (2D NOE) have been used to compare the conformational properties of 60-80 base pair long duplexes of the synthetic DNA polymer poly(dI-dC) with those of poly(dI-dbr5C) and poly(dG-dC) in the B and Z conformations. Cross peaks in the 2D NOE spectra arising from proton-proton dipolar interactions which are more or less independent of the DNA conformation are used to assign the spectra of these molecules. Other cross peaks are sensitive to the conformational details, and these are used to make deductions about the average conformation in solution. The proton-proton interactions that give rise to the cross peaks in the 2D NOE spectrum of poly(dI-dC) are indicative of a B family conformation and rule out the possibility of some alternative conformations, including A, Z, alternating B, and left-handed B-DNA. The spectra are similar to those obtained from B-form poly(dI-dbr5C) and poly(dG-dC) but different from Z-form poly(dG-dC). Taken together, these results indicate that the solution conformation of poly(dI-dC) is not unusual but more closely resembles that of other B-form DNAs.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

Experimental and calculated study of the vibrational modes of poly(dG-dC).poly(dG-dC) in B and Z conformations

Infrared spectra of the B and Z forms of poly(dG-dC).poly(dG-dC) are presented. Experimental assignments relative to certain vibration modes have been confirmed by calculation based on the GF-Wilson method. The calculated results show that only the geometry change between B and Z forms, is responsible for the observed modifications in the vibrational spectra.     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

Conformation of 145 base pair length poly (dG-dC) . poly (dG-dC) in solution and in association with histones.

We have studied the conformation of poly (dG-dC) . poly (dG-dC) in three conditions; i) associated with histones octamers, ii) alone at ionic strength 0.1, and ii) in solutions of over 2.5 M NaCl. The circular dichroism spectrum for the polymer bound to histones differs from that for the free polymer; the difference spectrum is similar to those for native and poly (dA-dT) . poly (dA-dT) core particles. Under the first two conditions, the 31P NMR spectrum is symmetric with line widths of 91 and 41 Hz, respectively, at 109.3 MHz. In high salt, two 31P peaks of equal intensity are observed, confirming recent results of Patel et al. (1) and indicating an alternating geometry for the phosphodiester backbone. Using this highly homogeneous DNA, we confirm that the Pohl-Jovin transition (2) is an intramolecular rearrangement, not requiring complete strand separation.