DNA Moiety Research Articles

Subsidiary hydrogen bonding of intercalated anthraquinonic anticancer drugs to DNA phosphate

Several anthraquinone derivatives are active against different kinds of human cancer. The cancerostatic activity has been mainly attributed to their ability to bind strongly to DNA by intercalation. Here, infrared spectroscopy was used to detect further, more specific DNA interactions with the prominent anticancer drugs daunomycin, adriamycin, aclacinomycin A and mitoxantrone as well as with the cytotoxic violamycin BI. The most striking result was a significant decrease in wave number of the band arising from antisymmetric stretching vibration of the PO 2 − groups of DNA upon complexation with adriamycin, aclacinomycin A, violamycin BI and mitoxantrone. This became evident after separation of the contributions from conformational changes of DNA to the influence on the wave number of that band. The drug-induced shift was interpreted in terms of the formation of a hydrogen bond between the intercalated drug molecules and the PO 2 − moiety of DNA via the following terminal hydroxyl groups: C14-OH for adriamycin, C4-OH for both aclacinomycin A and violamycin BI and, more tentatively, the external side- chain OH of mitoxantrone. Theoretical considerations, consisting of semi-empirical CNDO/2 calculations as well as normal coordinate analyses performed with molecular model fragments, provided results confirming and rationalising the experimental findings. The capacities of the anthracyclines for restriction of the conformational flexibility of DNA differ, presumably due to variations in the spatial dimensions of the sugar moieties of the drugs. The compatibility of the present results with data obtained from current geometrical models, especially those for the DNA-daunomycin and DNA-adriamycin complexes, is discussed in detail.

Purine salvage and metabolism inBabesia bovis

Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms of Babesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells by B. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.

Oxidative strand scission of DNA

Abstract

Reconstruction of the macromolecular antitumor antibiotic SN-07 from SN-07 chromophore and DNA.

To characterize the novel macromolecular antitumor antibiotic SN-07 and the role of DNA in SN-07, we reconstructed SN-07 in vitro from SN-07 chromophore and calf thymus DNA. Reconstructed SN-07 was identical with the native SN-07 in the ultraviolet absorption spectrum, nuclease-resistance characteristics, and biological activities (antibacterial activity and cytotoxicity). The DNA moiety promoted the cytotoxic activity of SN-07 chromophore against KB and HeLa cells, but suppressed the activity against some bacteria and L1210 cells. DNA containing guanine suppressed the activity more strongly than DNA not containing guanine against some bacteria and L1210 cells. The antibacterial activity of SN-07 chromophore was affected by dosage only when DNA was supplied, but not RNA or BSA. Thus the DNA composition of the SN-07 chromophore-DNA complex affected the activity of SN-07 chromophore and guanine had an important role of the binding of SN-07 chromophore to DNA.

Interaction of MgATP 2− with DNA: Assessment of metal binding sites and DNA conformations by spectroscopic and thermal denaturation studies

Spectroscopic (IR, UV, CD and fluorescence) and thermal denaturation studies of native calf thymus DNA, DNAMgATP 2− and DNAMg 2+ have been carried out in aqueous KBr medium (introduced by the present authors as a very effective solvent for DNA). The IR data recorded for the systems indicate that MgATP 2− binds to the N 7 and C 6O of the guanine residue of DNA forming a five-membered chelate ring. The data also suggest that despite binding to the guanine bases, Mg 2+ binds more strongly to the phosphate moiety of DNA. Solution CD spectra of DNA, DNAMgATP 2− and DNAMg 2+ indicate that in each case DNA exists in the B conformation. Thin-film CD studies reveal that irrespective of the relative humidity conditions, pure DNA as well as that after interaction with Mg 2+ show a structural transition B → C, conformationally, although belonging to the B family. A similar study shows that DNA on interaction with MgATP 2− assumes a more packed conformation (B) n giving rise to a ψ − spectrum. Steady-state as well as dynamic fluorimetric studies clearly indicate that MgATP 2− does not intercalate between CGGC base pairs. The thermal denaturation studies support the IR data with respect to the metal binding sites and the mode of binding in both cases.

Spin trapping of precursors of thymine damage in X-irradiated DNA.

A spin-trapping method combined with ESR spectroscopy was utilized to obtain evidence for the presence of precursor radicals leading to damage in X-irradiated DNA. Two technical improvements were introduced to the conventional spin-trapping method to make possible its application to large molecules such as DNA: prior to X irradiation, sonolysis of aqueous DNA solution by 19.5-kHz ultrasound was made to get a highly concentrated DNA solution and to lower the viscosity of the solution; after precursor radicals in X-irradiated DNA were trapped by a spin-trapping reagent, the DNA was digested to oligonucleotides by DNase I to get an ESR spectrum with a well-resolved hyperfine structure. Thus, it was recognized that the ESR spectrum obtained after X irradiation of the aqueous solution containing DNA and the nitroso spin-trapping reagent 2-methyl-2-nitrosopropane consisted of at least three sets of signals in the DNA. Identification of free radicals was made by comparing the spectrum with that of thymidine, which was precisely examined by a spin-trapping method combining two kinds of spin traps (nitroso and nitrone compounds) with liquid chromatography. As a result, all the signals were identified as the spin adducts of radicals produced at the thymine base moiety of DNA. The 5-hydroxy-5,6-dihydrothymin-6-yl radical was identified as a precursor of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), the 6-hydroxy-5,6-dihydrothymin-5-yl radical as a precursor of 6-hydroxy-5,6-dihydrothymine, and the 5-methyleneuracil radical as a precursor of 5-(hydroxymethyl) uracil.

Oxygen radical formation and DNA damage due to enzymatic reduction of bleomycin-Fe(III)

Aerobic incubations of bleomycin, FeCl3, DNA, NADPH, and isolated liver microsomal NADPH-cytochrome P-450 reductase resulted in NADPH and oxygen consumption and malondialdehyde formation, indicating that the deoxyribose moiety of DNA was split. All parameters measured depended on the active enzyme, bleomycin and FeCl3. In the absence of oxygen malondialdehyde formation was very low. When bleomycin, FeCl3 and the reductase were incubated with methional ethene (ethylene) was formed, suggesting that during the enzyme-catalyzed redox cycle of bleomycin-Fe(III/II) hydroxyl radicals were formed. Ethene formation also depended on oxygen, NADPH, the enzyme, bleomycin, and FeCl3. During aerobic incubations of bleomycin, FeCl3, NADPH, and isolated liver nuclei oxygen and NADPH were consumed and malondialdehyde was formed. Oxygen and NADPH consumption and malondialdehyde formation depended on bleomycin and FeCl3. In the absence of oxygen malondialdehyde was not formed. These results indicate that nuclear NADPH-cytochrome P-450 reductase redox cycles the bleomycin-Fe(III/II) complex and that the reduced complex activates oxygen, whereby hydroxyl radicals are formed which damage the deoxyribose of nuclear DNA.

A simple and convenient biochemical method for sex identification in the marine mussel, Mytilus edulis L.

One of two chromophores is formed on heating the mantle tissue of Mytilus edulis with thiobarbituric acid (TBA). Application of the test to male mussels yields a strong yellow colour ( λ max 453 and 490 nm), whereas in females, a pink colour ( λ max 532 nm) develops. While the latter is characteristic of the products of lipid peroxidation, it appears that the yellow colour may be derived from the 2-deoxyribose moiety of DNA. The TBA reaction can be used for the rapid, accurate sex identification in Mytilus edulis over 9–10 months of the year.

A sensitive colorimetric detection of virus DNA and oncogene

Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe. High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA. A highly sensitive colorimetric detection of virus DNA and oncogene was developed.

The reverse of the 'repair' reaction of thiols: H-abstraction at carbon by thiyl radicals.

Thiyl radicals (RS) formed by the reaction of radiolytically generated OH radicals with thiols, e.g. 1,4-dithiothreitol (DTT), react with cis- and trans-2,5-dimethyltetrahydrofuran by abstracting an H atom in the alpha-position to the ether function (k approximately equal to 5 X 10(3) dm3 mol-1 s-1). The so-formed planar ether radical is 'repaired' by the thiol (k = 6 X 10(8) dm3 mol-1 s-1) thereby regenerating a cis- or trans-2,5-dimethyltetrahydrofuran molecule. In this reaction a thiyl radical is reproduced. Thus trans-2,5-Me2THF from cis-2,5-Me2THF and vice versa are formed in a chain reaction: at a dose rate of 2.8 X 10(-3) Gys-1 and a trans-2,5-Me2THF concentration of 1 X 10(-2) mol dm-3 using DTT as the thiol, G(cis-2,5-Me2THF) = 160 has been found. The chain reaction is very sensitive to impurities and also to disulphides such as those radiolytically formed. 2,5-Me2THF can be regarded as a model for the sugar moiety of DNA where the C(4')-radical is known to lead to DNA strand breakage. The possible role of cellular thiols in the repair of the C(4') DNA radical, and also the conceivable role of thiyl radicals inducing DNA strand breakage, are discussed.

Reassembly of nucleosomal histone octamers during replication of chromatin.

We have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were density-labeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of single-stranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-substituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.

Current aspects on the radiation induced base damage in DNA.

In this short review, some current aspects of our knowledge about base damage in DNA induced by ionizing radiation will be summarized. It is not intended, to describe all the literature in this field; a very extensive review has been given in the book of Hüttermann et al. (1978) and also in later by Cadet and Berger (1985), Hutchinson (1985) and v. Sonntag and Schuchmann (1986). However, in this review, current ideas and unsolved problems concerning DNA base damage will be discussed, which may outline possible future research in this field. The understanding of DNA base damage requires the analysis of radicals formed in irradiated single DNA moieties as well as in whole DNA. Chemical studies about can be used for the molecular alterations of bases and biochemical methods for DNA-sequencing. In addition enzymes recognizing DNA damage and immunological methods with specific antibodies can be employed. However special emphasis should be given to the analysis of DNA base damage in irradiated cells and it will be shown, that a distinct gap in knowledge exists in this field in contrast to the radiation chemistry in aqueous solutions of DNA.

The interaction of the ruthenium(III)-chloride system with DNA

The interaction of RuCl 3 in aqueous solution with DNA has been studied at various r values. Electronic spectra, melting curves and sedimentation experiments indicate that Ru(III) is bound mainly to the phosphate moieties of DNA, causing stabilization of the double helix. For small values of r we observe renaturation upon cooling and possible interstrand cross-linking that persists at room temperature. During the second heating the melting temperature decreases, indicating substantial interaction of Ru(III) with the bases of the DNA. Ru(III) interacts with the nitrogen of the bases only when DNA is almost denaturated, and this cross-linking interaction seems to be quite strong.

The mechanisms of interaction between furanochromones and DNA. A heteronuclear Overhauser effect study on the khellin-thymidine model system.

The furanochromones khellin and visnagin have been characterised by 1H and 13C mono- and bidimensional NMR spectroscopies. Their strong affinity with DNA was experimentally confirmed by the complete disappearance of the furano-chromones' NMR signals upon additions of DNA. An intermolecular interaction between furanochromones and the thymidyl moieties of DNA, stabilized by the formation of a hydrogen bond between the thymidyl NH hydrogen and the C = O group of khellin or visnagin, is here proposed. This is suggested by the strong donor-acceptor behavior of these two molecular moieties, as pointed out by a selective 1H-13C Overhauser effect study of the khellin-thymidine model system.

ChemInform Abstract: Binding of Indium, Used in the Perturbed γ‐γ Angular Correlation Studies, on DNA and DNA Moieties.

AbstractThe two successive γ rays emitted from bound indium (IIIIn) are utilized in the title (PAC) method for the study of molecular dynamics of cellular macromolecules such as DNA.

Detection of DNA damage in human cells and tissues using sequencing techniques

Methods have been developed that permit both identification and location of sites of alterations is defined DNA sequences. These methods can be extended to human tissues using the alphoid segment, which comprises 1% of the human genome. This segment can be isolated in ample quantities from human cells and tissues. Once purified and end labeled, this defined segment can be used to detect sites of altered DNA moieties by combining Maxam-Gilbert sequencing protocols with appropriate enzymatic probes and chemical techniques. These studies can be performed in cultured cells or in tissues obtained by surgical excision or autopsy.

Binding of indium, used in the perturbed γ-γ angular correlation studies, on DNA and DNA moieties

The two successive gamma rays emitted from bound indium ( 111In) are utilized in the Perturbed γ-γ Angular Correlation (PAC) method for the study of the molecular dynamics of cellular macromolecules such as DNA. In this paper, evidence is presented indicating that indium binds on both phosphate and the base moieties of DNA and of the nucleotides and that, the binding of indium, under the conditions used in the PAC method, does not alter the hydrodynamic properties of the DNA macromolecule or its UV absorbance spectrum.

The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.

The microcomputer in cell and neurobiology research: edited by R. Ranney Mize, Elsevier, 1985. $49.50 (xv + 481 pages) ISBN 0 444 00842 X

For site-specific covalent labeling of oligodeoxynucleotides with redox probes, three general synthetic strategies are used. The first method introduces the probe phosphoramidite monomer during DNA synthesis. This phosphoramidite approach is amenable to derivatization of an oligodeoxynucleotide at the base 2'-deoxyribose, and at the phosphate. The second method, termed direct incorporation, involves labeling the solid support-bound oligodeoxynucleotide by palladium (Pd)-catalyzed cross-coupling chemistry. The chapter discusses the two approaches. Using the procedures described in the chapter, redox probes can be covalently attached to the nucleobase, ribose, or phosphate moiety of DNA. The synthetic procedures are amenable to both inorganic and organic redox probes. The site-specifically labeled ssDNA and dsDNA can be characterized by a number of techniques including mass spectroscopy, gel electrophoresis, high-performance liquid chromatography (HPLC), melting temperature, CD, and DSC to obtain information about identity, purity, duplex solution structure, and duplex thermodynamics. Redox probe-labeled oligodeoxynucleotides provide a unique means to systematically study charge-transfer reactions in DNA.

Model studies for the direct effect of high-energy irradiation on DNA. Mechanism of strand break formation induced by laser photoionization of poly U in aqueous solution.

Laser flash photolysis of polyuridylic acid (poly U) in anoxic aqueous solutions leads to biphotonic photoionization of the uracil moiety followed by the formation of single strand breaks (ssb). The rate constant for ssb formation (1.0 s-1, obtained from the slow component of conductivity increase at 23 degrees C and pH 6.8) increases with decreasing pH to 235 s-1 at pH 3.5. The activation energy (pre-exponential factor) was measured to be 66 kJ mol-1 (5 X 10(11) s-1) at pH 6.8. Addition of dithiothreitol (DTT) or glutathione (GSH) prevents ssb formation by reacting with a poly U intermediate (rate constant = 1.2 X 10(6) and 0.16 X 10(6) dm3 mol-1 s-1, respectively). Since with OH radicals as initiators very similar data have been obtained for the kinetics of ssb formation and for the reaction with DTT, we conclude that photoionization of the uracil moiety in poly U leads eventually to the same chemical pathway for ssb formation as that induced by OH radicals. Furthermore, we propose that protection by DTT and GSH occurs via H donation to the C-4' radicals of the sugar moiety of DNA and to the C-4' and the C-2' radicals of poly U.