Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution.

The decadeoxynucleotide d(AAAAATTTTT)2 in duplex form and the double-helical polynucleotide poly(dA).poly(dT) have been studied by Raman and infrared (IR) spectroscopy under a variety of environmental conditions. The IR spectra have been taken of cast films and compared to the IR spectra of the alternating poly(dA-dT), which shows clear B-genus and A-genus vibrational spectra under conditions of high (greater than 92%) and low (75%) relative humidity (RH). From the IR data, it is shown that d-(AAAAATTTTT)2 and poly(dA).poly(dT) adopt a B-genus conformation in films with high water content. When the relative humidity of the film is decreased, the IR spectra reflect a gradual evolution of the geometry of both d(AAAAATTTTT)2 and poly(dA).poly(dT) into a form intermediate between the B genus and A genus, but the IR spectrum of a pure A genus has not been obtained. In these DNAs at 75% RH, the IR bands of adenosine have the same frequencies as those found in poly(dA-dT) at 75% RH where the local furanose conformation is C3' endo/anti, but the thymidine frequencies do not resemble those of poly(dA-dT) at 75% RH but rather those of poly(dA-dT) at high humidities. It is concluded that both poly(dA).poly(dT) and d(AAAAATTTTT)2 adopt a fully heteronomous duplex geometry in cast films at low humidity. For studies in aqueous solution the Raman effect was employed. As a model for the heteronomous conformation in solution, the duplex poly(rA).poly(dT) was used.(ABSTRACT TRUNCATED AT 250 WORDS)

On the structure of poly d(A-S4T).

The helical twist of poly d(A-s4T) was determined from the periodicity of the cleavage patterns of the double stranded polydeoxynucleotide adsorbed on calcium phosphate and found to be 14 bp per turn. Both cleavage patterns and 31P NMR spectra indicate a mononucleotide structure rather than an alternating B DNA like poly d(A-T). The failure of nucleosome formation excludes a B type structure. The discrepancy of the mononucleotide structure found in 31P NMR spectra and the dinucleotide structure given by X ray fiber diffraction is explained by an alternating tilt of the planes of the base pairs (base roll) as a consequence of a strong propeller twist. The importance of interstrand stacking interactions of adjacent 4-thiothymidines for the helical stability is discussed.

Formation and removal of DNA adducts in rat liver treated with N-hydroxy derivatives of 2-acetylaminofluorene, 4-acetylaminobiphenyl, and 2-acetylaminophenanthrene.

Formation and removal of hepatic DNA adducts was studied in male Sprague-Dawley rats following single injections of two hepatocarcinogens, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and a nonhepatocarcinogen, N-hydroxy-2-acetylaminophenanthrene (N-OH-AAP) at 0.5 h, 1.5 h, 4 h, 24 h, 9 d and 29 d. Using a previously described 32P-postlabeling assay, maximal DNA binding of these compounds was observed at approximately 1.5 h, 0.5 h and 24 h, respectively. In addition to the formation of three already known C8- and N2-acetylated and C8-deacetylated guanine derivatives and several minor unknown adducts with N-OH-AAF, a set of four new major adducts was also detected. These comprised approximately 50% of the total adducts during the first 4 h. The three known adducts amounted to 58, 16 and 6% of the 1.5-h value after 24 h, 9 d and 29 d, respectively, while the bulk (greater than 84%) of the new major adducts were removed from the DNA within 24 h and found only in traces after 9 d. N-OH-AABP formed several unknown minor adducts, in addition to the one major C8-deacetylated and two minor C8- and N2-acetylated guanine derivatives; only the C8-deacetylated and N2-acetylated adducts were detected after 29 d. In the case of N-OH-AAP, two major and several minor adducts were detected, most of which were found to be deacetylated, and as much as 60% of the adducts measured at 24 h were still present after 9 d treatment. These data indicate that certain DNA adducts are repaired rapidly, while others persist for long periods.

A hydrogen exchange study of the open segment in a DNA double helix.

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly[d(A-T)] X poly[d(A-T)], poly(dA) X poly(dT), and constituent nucleosides (deoxyadenosine and thymidine) have been examined at various temperatures by stopped-flow ultraviolet spectrophotometry, in the spectral region 240-300 nm. The results were interpreted on the basis of a mechanism of the hydrogen exchange reaction of a helical polynucleotide, proposed by Englander and colleagues as well as by the Tsuboi and Nakanishi group. It was concluded that the rates of the base-pair opening reactions are nearly equal to one another in double-helical DNAs, irrespective of the base sequence. On the other hand, the free energy required for bringing the open segment at a particular base-pair was found to be much greater for poly(dA) X poly(dT) than for poly[d(A-T)] X poly[d(A-T)].

[Resonance Raman spectroscopy of polynucleotides]. / Spectroscopie Raman de résonance des polynucléotides.

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.

Intercalative and nonintercalative binding of large cationic porphyrin ligands to polynucleotides.

The interactions of two positional isomers and one analogue of meso-tetra (4-N-methylpyridyl) porphine, with the synthetic polynucleotides poly[d(A-T)] . poly[d(A-T)] and poly[d(G-C)] . poly[d(G-C)] have been investigated by circular dichroism. All four porphyrins were found to bind to the polynucleotides as shown by the induction of circular dichroism in their Soret bands. Furthermore, the sign of the induced ellipticity reflects selective occupation of binding sites by the porphyrin ligands. The conformational lability of poly[d(A-T)] X poly[d(A-T)] was found to be appreciable as micromolar amounts of meso-substituted 4-N-methylpyridyl, 3-N-methylpyridyl, and p-N-trimethylanilinium porphines induced a CD spectrum similar but not identical to that of DNA in the Z-form, i.e. a negative band at 280 nm and a positive band at 259 nm. The effect of porphyrin binding to poly[d(G-C)] X poly[d(G-C)] was less pronounced and dissimilar to that seen in the AT polymer.

Assignment of resonances in the phosphorus-31 nuclear magnetic resonance spectrum of poly[d(A-T)] from phosphorothioate substitution.

Two phosphorothioate analogues of poly[d(A$-T)] have been synthesized enzymatically. In one, poly[d(A$-T)], dTMP is replaced by thymidine 5'-O-phosphorothioate; in the other, poly[d(T$-A)], dAMP is replaced by 2'-deoxyadenosine 5'-O-phosphorothioate. The 31P NMR spectrum of poly[d-(A-T)] in solutions at low salt concentration shows two resonances at 51.80 and -4.25 ppm relative to trimethyl phosphate. The corresponding values for poly[d(T$-A)] are 51.51 and -4.43 ppm. These data allow the assignment of the downfield resonance at -4.23 ppm in poly[d(A-T)] to the phosphate group of d(TpA) and the resonance at -4.41 ppm to that of d(ApT). Thus, strong evidence is provided for a repeating dinucleotide structure. A comparison of the 31P NMR spectra of the various polymers in solutions of 2 M CsF reveals that both resonances are shifted upfield by approximately 0.9 ppm in the case of the phosphorothioates and by 0.2 or 0.4 ppm in the case of the phosphates. An upfield shift of about 0.18 ppm can also be observed for the two corresponding dinucleoside monophosphates. Thus, the upfield shift induced by high concentrations of CsF is not specific for the polymer backbone.

Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.

O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.

Salt-induced conformational transition of poly[d(A-T)] X poly[d(A-T)].

Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin.

The interaction between the B-form specific ligands netropsin (Nt) and distamycin-3 (Dst-3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non-B DNA form, putatively a Z-like form. Three polymers with strict alternating purine-pyrimidine sequences and GC content from 100-0% have been tested: poly(dG-dC) . poly(dG-dC), poly(dA-dC) . poly(dG-dT) and poly(dA-dT) . poly(dA-dT). The titrations by Nt and Dst-3 were followed by circular dichroism. Although specific binding of Nt to the Z-form of poly(dG-dC) . poly(dG-dC) does not occur, Nt reverses this Z structure to the B-type conformation; Dst-3 is, however, totally inefficient. The presumed non-B or Z-like structure of poly(dA-dC) . poly(dG-dT) is reversed to the B-form upon interaction with Nt; Dst-3 also induces this reversal but at higher ligand ratios. The modified B-structure of poly(dA-dT) . poly(dA-dT) in low water activity is efficiently reversed to the B-form by interaction with both Nt and Dst-3.