Thermal melting of poly(dA-dT).poly(dA-dT) in methanol-water solutions

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic analgetic used by drug abusers as a synthetic heroin, causes Parkinsonian symptoms in humans and degeneration of the substantia nigra in monkeys. MPTP is oxidized by brain mitochondrial preparations in a process which is blocked by deprenyl and pargyline, implying catalysis by monoamine oxidase B. The present paper demonstrates that pure MAO B isolated from beef liver oxidizes MPTP 38% as fast as benzylamine with a comparable Km value. Additionally, MAO A, isolated from human placenta, oxidizes MPTP to the same product at about 12% of the rate of kynuramine, again with a comparable Km value. The latter reaction is blocked by clorgyline. Both forms of MAO are progressively inactivated by MPTP by a process which follows first order kinetics. This progressive inactivation and the fact that the activity of MAO B is not significantly regenerated following gel exclusion chromatography suggest the formation of a covalent adduct with enzyme. Thus, MPTP appears to be a suicide inactivator of MAO.     

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.     

Titration of poly(dA-dT) . poly(dA-dT) in solution at variable NaCl concentration

CD and uv absorption data showed that high molecular weight poly(dA-dT) . poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl]< or =0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH- ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments.     

B-X transition in synthetic and natural (dA-dT)n.(dA-dT)n sequences

AB-X transition of polyh(dA-dT).poly(dA-dT) was observed to occur in methanol-water mixtures with methanol concentrations higher than 50% in the presence of a specific combination of monovalent and divalent cations. In the presence of Na+, divalent cations induce denaturation of poly(dA-dT).poly(dA-dT) accompanied by condensation and/or aggregation, and effect similar to that observed previously with random sequence DNA (Votavová, Kucerová, Felsberg and Sponar, J. Biomol. Struct. Dyn. 4,477-489, 1986). In the presence of Cs+ cations a B-X transition was induced by addition of Ca2+ or Mn2+ but not Mg2+ or Ni2+ ions. Circular dichroism and ultraviolet spectroscopy demonstrate that the X conformation is a double stranded form of poly(dA-dT).poly(dA-dT) belonging presumably to the B family which, however has an altered base stacking. The X conformation of poly(dA-dT).poly(dA-dT) found in methanol-water mixtures is a condensed and/or aggregated form. In contrast, the X conformation characterized by similar CD spectra observed in high salt concentrations is not aggregated up to a concentration of 6 M CsF. In methanol-water mixtures (A+T)-rich bacterial DNA behaves essentially as a random sequence DNA revealing no detectable amount of the X form. On the other hand crab (Cancer pagurus) satellite and crab non-satellite DNAs containing varying amounts of (dA-dT)n.(dA-dT)n sequences were shown to undergo a B-X transition, at least partly, in both methanol-water mixtures and 6 M CsF solutions.     

NMR relaxation and the structure of a synthetic DNA poly(dA-dT).poly(dA-dT)

1H-1H and 31P-1H nuclear Overhauser effects and 31P NMR spin-lattice relaxation times were measured for a synthetic DNA poly(dA-dT).poly(dA-dT) in a low-salt aqueous solution. The results have shown that all bases in the double helix are anti-orientated with respect to deoxyribose residues and that the sugar-phosphate backbone has an alternating architecture.     

The Z-conformation of poly(dA-dT).poly(dA-dT) in solution as studied by ultraviolet resonance Raman spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

Salt-induced conformational changes of poly(dA-dT).

Conformational changes of poly(dA-dT) . poly(dA-dT) induced by increasing ionic strength were studied using CD spectroscopy. It was found that a pronounced noncooperative inversion of the long-wavelength part of the CD spectrum of poly(dA-dT) . poly(dA-dT) occurred at high concentrations of CsF in solution. It was suggested that a great difference between the geometries of the purine and pyrimidine residues in the helix was characteristic of the structure of poly(dA-dT) . poly(dA-dT) in concentrated CsF solutions.     

Strance double helix of poly(dA-dT) in high-salt solution

³¹P and ¹H NMR studies indicate that double stranded poly(dA-dT) adopts an unusual structure in high-CsF solution, in which the dA residues occur in a unique geometry. This structure is different from the high-salt form of poly(dG-dC).     

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.     

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.     

Fractionation of poly (dA-dT) by gel chromatography

A commercial sample of poly (dA-dT), a copolymer of 2′-deoxyribosyladenosine (dA) and 2′-deoxyribosylthymidine (dT) of perfectly alternating sequence, was fractionated by chromatography on Agarose gel. Paucidisperse fractions of different molecular size were obtained. The plot of log s⁰_20,w values shows a linear dependence on V_e. The buoyant densities of individual fractions do not differ over the molecular size range studied. On the other hand, the heat-induced hyperchromic effect was found to depend on molecular size below a certain limit, s⁰_20,w = 4.12 S.     

Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates

The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results.     

Conformational variability of poly(dA-dT).poly(dA-dT) and some other deoxyribonucleic acids includes a novel type of double helix

The article reviews data indicating that poly(dA-dT).poly(dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT).poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the 31P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the 31P NMR spectrum of the limiting alternating B conformation of poly(dA-dT).poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack. However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the 31P NMR resonance of poly(dA-dT).poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC).poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC).poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT).poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT).poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT).poly(dA-dT) X-DNA. It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.