A left-handed (Z) conformation of poly(dA-dC).poly(dG-dT) induced by polyamines.

Blocks of potential Z-DNA forming alternating purine-pyrimidine (APP) sequences are widely dispersed in native DNAs. We have studied the effects of naturally occurring polyamines on the conformation of a synthetic APP sequence, poly(dA-dC).poly(dG-dT) by circular dichroism spectroscopy. In the presence of micromolar concentrations of spermidine (125 microM) and spermine (16 microM), this polymer undergoes B to Z transition in low ionic strength (2 mM Na+) buffers. The concentration of polyamines required for B to Z transition increases with Na+ in the buffer and a straight line is obtained on plotting ln[Na+] vs. ln [spermidine 3+]. However, at concentrations of polyamines higher than those necessary to induce B to Z transition, Z-DNA converts to psi-DNA, an ordered, twisted, tight packing arrangement of the double helix. These results suggest a pathway for the transient formation of Z-DNA segments in vivo by interaction of the ubiquitous polyamines with naturally occurring blocks of APP sequences.     

A Z-like form of poly(dA-dC).poly(dG-dT) in solution?

Circular dichroism was used to study changes in conformation of poly(dA-dC).poly(dG-dT) caused by a high concentration of various monovalent salts. It was found that CsF induced the gradual appearance of a negative band in the long wavelength part of the CD spectrum of poly(dA-dC).poly(dG-dT), which might reflect a transition of this DNA toward a Z-like structure.     

Right-handed and left-handed helices of poly(dA-dC) X (dG-dT).

The secondary structures of poly(dA-dC) X (dG-dT) were studied using CD and IR spectroscopies. We give spectroscopic evidence of secondary structure transitions of poly(dA-dC) X (dG-dT) from a B to a Z-like helix, induced by transition metal ions (Ni2+) in presence of high concentrations of Cs+ and Na+. In the presence of Na+, the B in equilibrium Z transition occurs at any temperature, whereas premelting conditions are required in presence of Cs+. For these two alkali ions the Z-like form is only induced by Ni2+ ions through their specific interactions at N7 of purines, under conditions of low water activity due to the high alkali salt concentration. We also show that the CD spectrum obtained in presence of Cs+ ions and characterized by a negative band at 275 nm, cannot be interpreted in terms of Z-like left-handed helix but reflects a modified B right-handed helix.     

Raman spectroscopic elucidation of DNA backbone conformations for poly(dG-dT).poly(dA-dC) and poly(dA-dT).poly(dA-dT) in CsF solution

Raman spectra have been recorded for poly(dG-dT) · poly(dA-dC) and poly(dA-dT) · poly(dA-dT) in low salt and at high concentrations of CsF. Poly(dG-dT) · poly(dA-dC) shows no change in the 682-cm^?1 guanine mode, demonstrating the absence of the Z-structure at high salt. The 790-cm^?1 phosphodiester symmetric stretch, however, shifts up 5 cm^?1 in 4.3M CsF, suggesting a slight conformational change, associated with ion binding or hydration changes. Poly(dA-dT) · poly(dA-dT) shows an additional broad band at 816 cm^?1, attributed to the phosphodiester modes associated with the C3′-endo deoxyribose units in the alternating B-structure. In this case, both the 841- and the 816-cm^?1 asymmetric phosphodiester stretches, associated with the C2′- and C3′-endo units, shift down on addition of CsF in a sequential manner. Correlation of this sequence with that previously observed for the two ³¹P-nmr resonances, establishes that the phosphodiester stretching frequencies depend on the conformation of the 5′-sugar, and not on the 3′-sugar.     

Conformational transitions of poly(dA-dT)poly(dA-dT) in ethanolic solutions.

Examination of circular dichroic and phosphorus nuclear magnetic resonance spectra showed that poly(dA-dT)-poly(dA-dT) exhibited an ethanol-induced transition to the A form in an Na+ containing medium like natural DNAs. A mere replacement of the Na+ by Cs+ counterions meant that the polynucleotide was with a little cooperativity transformed into a novel conformation displaying a deep negative band in the long wavelength part of the CD spectrum. The presence of very low concentration of Cs2+ shifted the midpoint of the transition to a lower content of ethanol.     

Ultrapolymorphic DNA: B, A, Z, and Z* conformations of poly(dA-dC).poly(dG-dT)

The synthetic polynucleotide poly(dA-dC).poly(dG-dT) at low ionic strength is shown to undergo conformational changes in the presence of [tris(2-aminoethyl)amine]zinc(II) chloride (ZnN4). At 100 microM ZnN4, circular dichroism and 31P NMR spectra show the formation of Z DNA. With an increase of the concentration up to 600 microM, an A-like form is obtained, and at still higher concentration, the polynucleotide reverts to the original B form. Experiments on polynucleotide samples in which some sequence errors were observed showed that spermine was necessary as well as ZnN4 to induce the Z form. At higher concentrations of spermine and ZnN4, a second Z form (Z*) is observed. Raising the ionic strength inhibits the formation of the Z form, whereas the presence of ethylene glycol favors it.     

A twenty-two-fold increase in the relative affinity of estrogen receptor to poly (dA-dC).poly (dG-dT) in the presence of polyamines.

We studied the relative efficacy of polyamines to facilitate the binding of estrogen receptor to poly(dA-dC).poly(dG-dT). In the absence of polyamines, 1,400 micrograms/ml of this polynucleotide eluted 50% of bound estrogen receptor from DNA-cellulose. In contrast, 50% estrogen receptor was eluted by 65 micrograms/ml of poly(dA-dC).poly(dG-dT) complexed with 150 microM spermidine. Putrescine and spermine also enhanced the ability of poly(dA-dC).poly(dG-dT) to elute estrogen receptor, but the magnitude of the effect was not as high as that of spermidine. Control experiments with calf thymus DNA and poly(dA-dT).poly(dA-dT) showed 6- and 3-fold increase, respectively in their affinity for estrogen receptor in the presence of spermidine. The dramatic increase in the affinity of poly(dA-dC).poly(dG-dT) for estrogen receptor in the presence of polyamines might be a result of the conversion of the polynucleotide to the left-handed Z-DNA form. These results show that polyamines are capable of participating in estrogenic regulation of gene expression by altering the affinity of the receptor for specific DNA sequences.     

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

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

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Conformational transitions of poly(dA-bromo5dU) and poly(dA-iodo5dU) in solution.

Extensive circular dichroism studies have been conducted with the title polynucleotides under various solution conditions. The studies provided the following information: (i) The halogen atoms in place of thymine methyl hinder the isomerization into X-DNA. (ii) The brominated but not iodinated polynucleotide isomerizes into Z-DNA in concentrated NaCl+NiCl2. The transition takes place at lower NiCl2 concentrations than with poly(dA-dT). (iii) The iodinated polynucleotide forms an unusual conformation in aqueous solution in which it is very stable. It isomerizes from this conformer into the usual B-type double helix in concentrated ethanol solutions. The isomerization is a two-state cooperative process. (iv) Both title polynucleotides undergo still another two-state cooperative transition in trifluorethanol solutions presumably into A-DNA showing a rather unusual circular dichroism spectrum.     

Conformational stability of alternating d (CG) oligomers in high salt solution.

The conformation of d (CG)n oligomers with n = 2,3 has been studied in aqueous solution in the presence of high salt concentration. A minimum n value of three is necessary to obtain a left-handed Z-helix. When d (CG)3 is flanked by three non Z-helicogenic alternating AT sequences the left-handed helix is unstable and a B-type conformation is obtained also at high salt concentration.     

Conformational transitions of a synthetic DNA poly(dA-dU). poly(dA-dU) in concentrated solutions of caesium fluoride

Chiroptical properties of poly(dA-dU).poly(dA-dU) were studied in concentrated NaCl and CsF solutions to reveal the role of the alternating B conformation in the CsF-induced alternating B-X conformational transition of poly(dA-dT).poly(dA-dT). Poly(dA-dU).poly(dA-dU) has been chosen for this purpose because it has, instead of the alternating B conformation, a regular conformation like poly(dG-dC).poly(dG-dC) in low-salt solution. It was found that poly(dA-dU).poly(dA-dU) did not assume that Z form at high NaCl concentrations but exhibited extensive CsF-induced changes in the circular dichroism spectra like poly(dA-dT).poly(dA-dT). The changes of reflect two consecutive two-state conformational transitions of the polynucleotide, both taking place with fast kinetics and low cooperativity. The transition were interpreted as involving the regular and alternating B conformation at lower CsF concentrations and the alternating B and X conformation at higher CsF concentrations. A comparison of the behaviour of poly(dA-dU).poly(dA-dU) and poly(dA-dT).poly(dA-dT) in CsF solutions demonstrates that the thymine methyl groups promote the X form but are not crucial for its existence. On the other hand, the alternating B conformation appears to be the inevitable starting structure for DNA isomerization into the X form.     

Conformational states of poly(L-tyrosine) in aqueous solution.

Poly(L -tyrosine) [(L -Tyr)_n] has been characterized in aqueous solution using circular dichroism (CD) and infrared (ir) spectroscopy, and ultracentrifugal analysis. Most of the experiments were carried out at 0.01% polymer or less to avoid the complications caused by precipitation previously encountered by others. This permitted us to study solutions of (L -Tyr)_n at lower pH values than had been attained previously. Our results show that a transition to an antiparallel-β conformation occurs at pH 11.32 upon titration from higher pH. The β structure is intramolecular when first formed and aggregates with time or upon titration below pH 11. Ultracentrifugal analysis of the intramolecular β conformation shows that it is quite compact, with a frictional coefficient ratio, f/f_min, of 1.09. In addition to the β structure, a nonordered form of the polymer has been obtained below pH 11 by rapid titration of the ionized polyelectrolyte. This form is nonaggregated and was found to have an f/f_min of 1.01, and is therefore almost spherical. The aggregated β form was found to be thermodynamically more stable than the nonordered form at pH 10.7.     

Salt induced transitions between multiple conformations of poly (rG-m5dC).poly (rG-m5dC).

Salt induced transitions between four conformations of the methylated ribo-deoxyribo co-polymer poly (rG-m5dC).poly (rG-m5dC) have been studied using phosphorous-NMR, Raman spectroscopy, and circular dichroism. A high salt A-Z transition is observed for the polymer. However, the methylated polymer does not enter the high salt Z form more readily than the analogous unmethylated polymer, unlike the effect of methylation on the fully deoxy polymer poly (dG-dC).poly (dG-dC). The methylated polymer fails to undergo a low salt A-Z transition in 5 mM Tris buffer, unlike the unmethylated poly (rG-dC).poly (rG-dC). However, if the counterion is changed to triethanolamine buffer, an A-Z transition does take place. In 5 mM Tris buffer the phosphorous-NMR spectrum of poly (rG-m5dC).poly (rG-m5dC) shows one resonance in the absence of NaCl that splits into two closely spaced resonances as the NaCl level is increased to 30 mM. The Raman spectrum of poly (rG-m5dC).poly (rG-m5dC) shows that it is in the A conformation at intermediate salt concentrations. From this we conclude that poly (rG-m5dC).poly (rG-m5dC) is in a regular A conformation in Tris buffer at low Na+ levels, shifting to an alternating A conformation with a dinucleotide repeat at intermediate salt concentrations.     

Conformational transitions and aggregation in poly(dA)-poly(dT) system induced by Na+ and Mg2+ ions

Effects of Mg²⁺ ions on thermally induced conformational transitions in the synthetic poly(dA)·poly(dT) and poly(dA)·2poly(dT) were studied in the buffered solutions (pH 6.9), containing 0.1 or 1 M NaCl at polynucleotide concentration of 0.1–0.3 mM (in nucleic bases). The experiments consist of measurements of the UV absorption and intensity of conventional visible static light scattering. The diagram of conformational transitions in the poly(dA)–poly(dT)–Mg²⁺ system was constructed on a basis of experimental data obtained. Anomalously strong light scattering, like critical opalescence, has been revealed at 0.1 M NaCl and [Mg²⁺]≥20 mM in the melting range of both polynucleotides, which eventually disappeared after the completion of polymer strands separation. The effect presumably is caused by a fluctuation process of polymer strands complexing which arises at a certain concentration of Mg²⁺ ions.     

Conformational transitions and hydration of poly d (A-T) · poly d (A-T) in fibers

Conformational transitions of poly d(A-T) · poly d (A-T) have been studied by fiber X-ray diffraction and measurement of fiber dimensions. Results obtained for the D-A-B and D-B transitions are presented and analyzed. For all these form transitions, cooperativity effects are observed for the variation of the rise per nucleotide versus the relative humidity. Detailed information about hydration of the polynucleotide during form transitions and the numbers of water molecules per nucleotide necessary to stabilize the different helical conformations are presented. Offprint requests to: S. Premilat     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.