Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

[Astrobiological investigations on board Russian space crafts].

The possibility of prebiotic synthesis of nucleic acids components (nucleotides) has been demonstrated under condition of the space orbital stations and satellites under effect of all space radiation spectra. Since a lot of different nucleic acids components are known to be present within small space bodies, we have to investigate their chemical complication in respect with such components as nucleotides. The goal of this work is to review our results in the field of prebiotic synthesis of purine and pyrimidine nucleotides on the board of Russian space crafts. The increase in the solid reaction mixtures exposure time leads to degradation of both initial components (nucleosides) and the reaction products (nucleotides). The dominating role of heat energy in the abiogenic reactions has been revealed in laboratory (ground) experiments. Similar set of natural nucleotides has been synthesized under effect of different open space energy sources in both flight and ground experiments. The formation of 5'-nucleotides is a dominating process. All the data are discussed in the context of exobiological investigations on the Earth's orbit.

Phenyl radicals react with dinucleoside phosphates by addition to purine bases and H-atom abstraction from a sugar moiety.

Laser-induced acoustic desorption combined with mass spectrometry has been used to demonstrate that phenyl radicals can attack dinucleoside phosphates at both the sugar and base moieties, that purine bases are more susceptible to the attack than pyrimidine bases, and that the more electrophilic the radical, the more efficient the damage to dinucleoside phosphates.

Altered intracellular purine nucleotides in gamma-irradiated red blood cell concentrates.

BACKGROUND AND OBJECTIVES: Gamma irradiation at a dose of 30 Gy induces deterioration of erythrocytes, resulting in storage lesions that significantly shorten the shelf-life of packed red cell concentrates (RCCs). The aim of the present study was to investigate the effect of gamma irradiation on intracellular purine nucleotides of red blood cells during storage. MATERIALS AND METHODS: Three-day-old leucocyte-depleted saline-adenine-glucose-mannitol (SAGM)-preserved RCCs, obtained from the Blood Service of the Austrian Red Cross, were gamma irradiated with 30 Gy. Samples were taken on days 1, 2, 3 and 7 after irradiation and subsequently at weekly intervals up to the end the of shelf-life (day 39 after irradiation) and were investigated for the K+ and Na+ content in the supernatant, for intracellular concentrations of ATP, ADP, ITP, IDP, GTP and GDP of erythrocytes, and for haemolysis. RESULTS: Within the first 24 h after gamma irradiation, no metabolic or biochemical changes were detectable in the RCCs. The K+ concentration in the supernatant increased after 24 h, while the Na+ concentration decreased in irradiated units and this ion disequilibrium persisted until the end of the shelf-life. After an initial increase of intracellular ATP, ADP and GTP during the first week of storage, the intracellular concentrations of ATP, ADP, GTP and ITP decreased, while IDP increased. The decrease of ATP and ADP was found to be more pronounced in irradiated units. At the end of the shelf-life, the ATP, GTP and ITP concentrations of irradiated RCCs had decreased to < 10% of the initial level and the critical threshold of 0.8% haemolysis was reached. CONCLUSION: Gamma irradiation of SAGM-preserved RCCs leads to serious deterioration of the purine nucleotide metabolism of erythrocytes during storage, which can reduce the in vivo recovery of the transfused red cells.

Origin of ultraviolet damage in DNA.

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.

Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer. relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented.

[Modeling radiation damage to DNA using radionuclides incorporated into polynucleotides]. / Modelirovanie radiatsionnykh povrezhdenii DNK pri pomoshchi deistviia vkliuchennykh v polinukleotidy radionuklidov.

Because of a great variety and different reparability of radiation-induced DNA lesions it is difficult to evaluate the radiobiological significance of certain individual alterations. It is suggested that the radionuclides incorporated into DNA can be used to imitate different types of radiation damages to DNA. Both qualitative and quantitative aspects of the problem are discussed.

[Simulation of the effects of radiation injury of DNA and genetic hazards of radionuclide decay. Dehydration of purine nucleotides during decay of an incorporated tritium]. / Modelirovanie éffektov radiatsionnykh povrezhdenii DNK i geneticheskaia opasnost' raspadov radionuklidov. Degidrirovanie purinovykh nukleotidov pri raspade inkorporirovannogo tritiia.

The dehydrogenation of purine nucleotides in position 8 is not a severe lethal injury. In addition, the dehydrogenation in the eight position of adenine is not an effective mutagenic event. The dehydrogenation in the eight position of guanine is a mutagenic damage. As to the induction of point mutations, 3H is not more hazardous than external gamma-radiation delivered in equivalent doses.