2--deoxyguanosine-5--phosphate and 2--deoxy-5--adenosine-monophosphate

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Topics: Adsorption; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA; Nanopores; Nanotechnology; Spectrum Analysis, Raman

Density functional theory has been used to investigate the oxidation of a guanine nucleotide by platinum(IV), a process that can be important in the degradation of DNA. For the first time, we have provided a comprehensive mechanism for all of the steps in this process. A number of intermediates are predicted to occur but with short lifetimes that would make them difficult to observe experimentally. A key step in the mechanism is electron transfer from guanine to platinum(IV), and we show that this is driven by the loss of a chloride ligand from the platinum complex after nucleophilic attack of 5'-phosphate to C8 of guanine. We have investigated several different initial platinum(IV) guanine adducts and shown that the adduct formed from replacement of an axial chlorine ligand in the platinum(IV) complex undergoes oxidation more easily. We have studied adenine versus guanine adducts, and our results show that oxidation of the former is more difficult because of disruption of the aromatic π system that occurs during the process. Finally, our results show that the acidic hydrolysis step to form the final oxidized product occurs readily via an initial protonation of N7 of the guanine.

Topics: Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Fragmentation; Electron Transport; Ligands; Models, Chemical; Models, Molecular; Oxidation-Reduction; Platinum; Protons; Quantum Theory

Continuous oxidative damage inflicted on DNA produces 7,8-dihydro-8-oxoguanine (8-oxoG), a commonly occurring lesion that can potentially cause cancer by producing G → T transversions during DNA replication. Mild oxidation of 8-oxoG leads to the formation of hydantoins, specifically guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), which are 100% mutagenic because they encode almost exclusively the insertion of dAMP and dGMP (encoding G → T and G → C transversions, respectively). The wild-type (wt) pol α family DNA polymerase from bacteriophage RB69 (RB69pol) inserts dAMP and dGMP with low efficiency when situated opposite Gh. In contrast, the RB69pol Y567A mutant inserts both of these dNMPs opposite Gh with >100-fold higher efficiency than wt. We now report the crystal structure of the "closed" preinsertion complex for the Y567A mutant with dATP opposite a templating Gh (R-configuration) in a 13/18mer primer-template (P/T) at 2.0 Å resolution. The structure data reveal that the Y to A substitution provides the nascent base pair binding pocket (NBP) with the flexibility to accommodate Gh by allowing G568 to move in the major-to-minor groove direction of the P/T. Thus, Gh is rejected as a templating base by wt RB69pol because G568 is inflexible, preventing Gh from pairing with the incoming dATP or dGTP base.

Topics: Amino Acid Substitution; Bacteriophages; Crystallography, X-Ray; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA-Directed DNA Polymerase; Guanidines; Hydantoins; Models, Molecular; Viral Proteins

Steady-state and time-resolved studies of the fluorescence of four aromatic unconjugated pterins (pterin (Ptr), 6-(hydroxymethyl)pterin (Hmp), 6-methylpterin (Mep), and 6,7-dimethylpterin (Dmp)) in aqueous solutions in the presence of different nucleotides (2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-monophosphate (dAMP), and 2'-deoxycytosine 5'-monophosphate (dCMP)) have been performed using the single-photon counting technique. The singlet excited states of acid forms of pterins are deactivated by purine nucleotides (dGMP and dAMP) via a combination of dynamic and static processes. The efficiency of the dynamic quenching is high, independently of the nature of the purine base of the nucleotide and of the chemical structure of the substituents linked to the pterin moiety. Analysis of the static quenching indicates that ground-state association between pterins and purine nucleotides takes place, but the formation of the corresponding complexes is significant only at relatively high reactant concentrations. The quenching of the fluorescence of acid forms of pterin derivatives by dCMP, a pyrimidine nucleotide, is slightly less efficient than the quenching by purine nucleotides and is purely dynamic. In alkaline media, the fluorescence quenching is much less efficient than in acidic media, the deactivation by purine nucleotides being purely dynamic, whereas quenching by dCMP is negligible. Possible mechanisms for the quenching of fluorescence of pterin derivatives by the different nucleotides are discussed.

Topics: Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Deoxyribonucleotides; Fluorescence; Hydrogen-Ion Concentration; Kinetics; Molecular Structure; Pterins; Spectrometry, Fluorescence; Thermodynamics; Water

We previously reported the synthesis of a borononucleotide analogue of thymidine monophosphate and its association towards the formation of a new borono-linked dinucleotide. Here we describe the completion of the set of four 2'-deoxyborononucleotide analogues of natural nucleotide monophosphates, namely the previously unknown dCbn, dGbn and dAbn. These analogues were all prepared from the respective 5'-aldehydic nucleosides through a homologation/reduction sequence. The borononucleotides were subsequently obtained by either borylation (dCbn and dGbn) or cross-metathesis (CM) in the presence of the Hoveyda-Grubbs catalyst (dAbn). The reversible formation of the corresponding dinucleotides between these new analogues and uridine was studied by (1)H NMR, and semi-empirical calculations were carried out to provide bond length and electrostatic information that assess the structural similarities existing between these bioisosteres and their natural counterparts.

Topics: Boron; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Deoxyribonucleotides; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Uridine

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.

Topics: Circular Dichroism; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyguanosine; DNA; DNA Polymerase I; DNA Replication; Escherichia coli; Oligodeoxyribonucleotides; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Thermodynamics

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.

Topics: Boron Compounds; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; Electrophoresis, Capillary; Ethylenediamines; Fluorescent Dyes; Lasers; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Sulfites; Thymidine Monophosphate

Reactive oxygen species can give rise to intrastrand cross-link lesions, where two neighboring nucleobases are covalently bonded. Here, we employed LC-MS/MS and demonstrated for the first time that gamma irradiation of a synthetic duplex oligodeoxyribonucleotide can give rise to an intrastrand cross-link lesion G[8-5]C, where the C8 carbon atom of guanine and the C5 carbon atom of its 3'-neighboring cytosine are covalently bonded. We also carried out in vitro replication studies of a substrate containing a site-specifically incorporated G[8-5]C, and our results showed that yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion. Steady-state kinetic analyses for nucleotide incorporation by pol eta showed that the 3'-cytosine moiety of the cross-link did not significantly affect either the efficiency or the fidelity of nucleotide incorporation. The 5' guanine portion of the cross-link lesion, however, markedly reduced both the efficiency and the fidelity of nucleotide incorporation; the insertion of dGMP or dAMP was slightly favored over the insertion of the correct nucleotide, dCMP, which was in turn favored over the insertion of dTMP. The above results support that the oxidative cross-link lesion, if not repaired, can be mutagenic.

Topics: Chromatography, Liquid; Cytosine; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; Gamma Rays; Guanine; Mass Spectrometry; Nucleic Acid Heteroduplexes; Saccharomyces cerevisiae Proteins; Thymidine Monophosphate

The molecular structure of different conformers of isolated canonical purine 2'-deoxyribonucleotides 2-deoxyadenosine-5'-phosphate (pdA) and 2'-deoxyguanosine-5'-phosphate (pdG) was optimized using the B3LYP/6-31G(d) method. The results of the calculations reveal that the geometrical parameters and relative stability of the conformers significantly depend on the nature of the nucleobase, its orientation, the conformation of the furanose ring, the charge of the phosphate group and the character of the intramolecular hydrogen bonds. Analysis of the electron density distribution in purine nucleotides reveals the existence of a number of intramolecular hydrogen bonds. In general, the south conformer has a lower energy at the anti orientation of the base, and both conformers occur as the most stable for the syn orientation of the nucleobases. The results of the calculations reveal that the geometry and relative energy of the conformers of purine DNTs may be easily tuned by the charge of the phosphate group.

Topics: Anions; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Drug Stability; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; Purine Nucleotides; Pyrimidine Nucleotides; Thermodynamics

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Topics: Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Exonucleases; Female; Isotope Labeling; Micrococcal Nuclease; Monocrotaline; Phosphorus Radioisotopes; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Reproducibility of Results; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Mass, Electrospray Ionization

We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.

Topics: Borates; Buffers; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Electrophoresis, Capillary; Hydrogen-Ion Concentration; Polyethylene Glycols; Surface-Active Agents; Thymidine Monophosphate

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.

Topics: Adenosine Monophosphate; Antineoplastic Agents; Cytidine Monophosphate; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Dimethylformamide; Guanosine Monophosphate; Hydrogen-Ion Concentration; Magnesium; Magnetic Resonance Spectroscopy; Nucleotides; Organometallic Compounds; Solvents; Spectrophotometry, Infrared; Thymidine Monophosphate; Titanium

The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models, and its environmental presence, suggest its potential importance with regard to human cancer development. Depending on the bioassay model, 6-NC can be activated by simple nitro reduction, ring oxidation, or by a combination of ring oxidation and nitro reduction. Only the first pathway has been clearly established. Thus, this study purports to unequivocally define the other pathways. Toward this end, we report for the first time the synthesis of anti-1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-6-nitrochrysene (6-NCDE), a likely ultimate carcinogenic metabolite of 6-NC. Also, we describe our initial investigation of its binding with calf thymus DNA, 2'-deoxyguanosine-5'-monophosphate (2'-dGuo), and 2'-deoxyadenosine-5'-monophosphate (2'-dAdo) in vitro. These adduct markers were then employed for comparison with those obtained in the rat after in vivo treatment with 6-NC. On the basis of the results, it appears that the major adduct formed in the liver of rats treated with 6-NC is not derived from 6-NCDE.

Topics: Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Chrysenes; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Magnetic Resonance Imaging; Thymus Gland

To investigate the antioxidative activity of the constituents of the roots of Scrophularia ningpoensis (Chinese name: Xuanshen).. The main compounds from the roots of Scrophularia ningpoensis were isolated and identified by chromatography and FABMS, NMR etc. Using the techniques of pulse radiolysis, the electron transfers from iridoid glycosides (IG) or phenylpropanoid glycosides (PG) to oxidized OH radical adducts of 2'-deoxyadenosine-5'-monophosphate acid (dAMP) or 2'-deoxyguanosine-5'-monophosphate acid (dGMP) were observed.. Two IG: harpagoside and harpagide, two PG: angoroside C and acteoside were obtained as the main hydrophilic constituents of the plant. At 0.1 mmol/L concentration, angoroside C and acteoside were able to repair the oxidized OH adducts dAMP and dGMP significantly. However, harpagoside and harpagide had no such effect. The electron transfer rate constants of angoroside C with dAMP and dGMP were 4.2 x 10(8) and 10.3 x 10(8) L.mol-1.s-1; the electron transfer rate constants of acteoside with dAMP and dGMP were 5.3 x 10(8) and 20.2 x 10(8) L.mol-1.s-1.. PG from Scrophularia ningpoensis have a potent antioxidative activity for reducing of the oxidized OH adducts of dAMP and dGMP.

Topics: Antioxidants; Coumaric Acids; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Drugs, Chinese Herbal; Glucosides; Hydroxyl Radical; Phenols; Plants, Medicinal; Scrophulariaceae; Trisaccharides

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.

Topics: Animals; Cattle; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Damage; DNA Polymerase beta; DNA Polymerase I; DNA Replication; DNA-Directed DNA Polymerase; Guanosine; Humans; Hydantoins; Iridium; Mutagenesis; Nickel; Oxidants; Oxidation-Reduction

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.

Topics: Animals; Autoradiography; Benzopyrenes; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Epoxy Compounds; Exonucleases; Micrococcal Nuclease; Microsomes, Liver; Phosphorus Radioisotopes; Rats; Spectrometry, Fluorescence; Stereoisomerism

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

Topics: Alkylation; Animals; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; DNA Adducts; Mass Spectrometry; Melphalan; Sensitivity and Specificity; Thymidine Monophosphate

In this work radicals generated by dissociative electron attachment to iodoacetamide (H2NCOCH2.) and 6-chloromethyluracil (U5CH2.) are suggested to react with DNA nucleotides in frozen aqueous solutions via either hydrogen abstraction or addition to the double bonds of the bases. Methyl hydrogens of TMP are the preferential sites of the attack by H2NCOCH2. radical. For dCMP the C1' site on the sugar group is found to be the preferred site of hydrogen abstraction by H2NCOCH2. while for dGMP and to a lesser extent dAMP attack at the C8 position of the purine ring is found to be competitive with sugar attack. In general allylic U6CH2. and U5CH2. radicals are found to be poor hydrogen abstractors and the only reaction pathway found is the addition to double bonds at C6 in thymine and C8 in adenine and guanine. Whereas, the cytosine 5,6 double bond appears to be unreactive towards addition at low temperatures. Some evidence is found for sugar radical addition to the adenine C8 position.

Topics: Adenine; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; Electron Spin Resonance Spectroscopy; Free Radicals; Gamma Rays; Guanine; Iodoacetamide; Kinetics; Molecular Structure; Oligodeoxyribonucleotides; Thermodynamics; Thymidine Monophosphate; Thymine; Uracil

UV differential spectroscopy is applied to study the interaction of Cu2+, Ni2+, Mn2+ ions with deoxyribonucleotides of canonic bases (dGMP, dAMP, dCMP, dTMP) and native DNA. Heteroatoms of the bases, coordinating ions, and binding constants which characterize the formation of metal complexes are found. The affinity of the ions is lower for the deoxyribonucleotide bases than for the ribonucleotide ones. This indicates that 02' of ribose participates in the stabilization of the metal complex even under conditions close to the neutral one (pH 6). Unlike the Cu2+ ions, Ni2+ and Mn2+ ions do not interact with N3C both in monomers and polymers. This seems to be the main factor explaining why copper makes DNA transform into a structure with a quasi-Hoogsteen pairing of GC pairs. No transformations of this kind of helix-coil transitions are caused by manganese and nickel up to concentrations 4 X 10(-2) M.

Topics: Copper; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; Manganese; Molecular Structure; Nickel; Nucleic Acid Conformation; Ribonucleotides; Thymidine Monophosphate

An ESR investigation of the individual DNA base radicals produced by gamma-irradiation of frozen solutions of the nucleotides TMP, dCMP, dGMP and dAMP and their reactions with cysteamine upon annealing is reported. The results show that water radicals in bulk ice do not lead to the formation of DNA or cysteamine radicals. Radicals from the oxidation pathway which include the DNA base one electron oxidized radicals and their successors, G(C8)OH., A(C8)OH. and thymine dimers (.Tdi) and/or T(C6)OH; readily react with cysteamine to form RS. and ultimately RSSR-. Reactions of dGMP and dCMP radicals from the oxidation pathway with cysteamine occur at lower temperatures than those of dAMP and TMP, suggesting hole migration. Both T(C6)H. and C(N3)H. react with cysteamine to form RS. and diamagnetic products, but G(C8)H. and A(C8)H. do not. Subtraction of the anion radical T-. and its proton adduct T(C6)H. from the total radical yield of TMP (with or without cysteamine) suggests that somewhat less than half of the total TMP radicals found are a result of the oxidative pathway. Similar results are found in the other nucleotides. The total spectral intensity derived from the radicals from the oxidative pathway such as G(C8)OH., A(C8)OH. and .Tdi/T(C6)OH. are somewhat less than that for the protonated anion radicals. Only one non-base radical is identified, a sugar radical at the C(1)' site on the deoxyribose portion of dAMP. This species, S(A)., is also found to react with cysteamine or its disulfide radical anion. Analyses performed in the presence and absence of a thiol are found to allow for a clear separation of oxidative and reductive pathways.

Topics: Cysteamine; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA; Free Radicals; Freezing; Hydroxyl Radical; Nucleotides; Thymidine Monophosphate

We have identified a type of secondary structure for the homopurine oligomer d(A-G)10 below pH 6 in 0.01 M Na+ that is characterized by intense CD but only minor hypochromicity. The stability of this helix, designated d(A(+)-G)10, does not depend on oligomer concentration and increases sharply as ionic strength or pH drops, reaching a maximum at 4.0 (melting temperature, 37 degrees C). The pKa for the transition, 5.3 at 25 degrees C and even higher with decreasing temperature and [Na+], is much higher than the intrinsic pKa values for dA or dG residues. While the dA residues are protonated in the helix, further protonation of the dG residues disrupts it. When observed at 280 nm, melting of the helix first results in hypochromicity due to stacking of extrahelical dG residues with neighboring dA residues. The character and temperature dependence of the CD spectra of the constituent dinucleoside monophosphates indicate minimal chirality and base overlap for the A+pG sequences in d(A(+)-G)10 but left-handed twist with some base overlap for the GpA+ sequences. The observed properties are best satisfied by a model for an intramolecular helix with limited base overlap, stabilized by ionic bonds between dA residues protonated at N-1 and downstream negatively charged phosphates brought close due to the backbone helical twist, while Gsyn residues lie external to the helix. This structure could provide additional stabilizing energy for biologically relevant protonated non-B-DNA structures adopted by homopurine.homopyrimidine sequences due to topological stress or specific protein binding.

Topics: Circular Dichroism; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Hydrogen-Ion Concentration; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligodeoxyribonucleotides; Spectrophotometry, Ultraviolet; Thermodynamics

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.

Topics: Cyclic N-Oxides; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA; Electron Spin Resonance Spectroscopy; Pyrrolidines; Spin Labels; Thymidine Monophosphate

The degradation of the nucleotides dAMP, dGMP, dCMP and dTMP and of calf thymus DNA by ozone was studied. In all cases both base and sugar moiety were degraded. Furthermore, strand breaks were induced in calf thymus DNA. Hydroxyl radicals were probably involved in the oxidation of the base in dAMP and of the deoxyribose ring, but not in the degradation of the other bases. This indicates that ozone-induced DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals.

Topics: Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA Damage; Free Radicals; Hydroxides; Hydroxyl Radical; Ozone; Spectrophotometry, Ultraviolet; Thymidine Monophosphate

When Escherichia coli DNA polymerase I (Pol I) replicates a homopolymer, the excision/polymerization (exo/pol) ratio varies with enzyme and initiator concentration. The study of this effect in the case of poly(dA).oligo(dT) replication led us to propose a mnemonic model for Pol I, in which the 3' to 5' excision activity warms up when the enzyme is actively polymerizing, and cools down when it dissociates from the template. The model predicts that the exo/pol ratio must increase with processivity length and initiator concentration and decrease with enzyme concentration. It predicts also that contact of the enzyme with one template alters its excision efficiency towards another template. The exo/pol ratio and processivities of Pol I and its Klenow fragment were studied on four templates: poly(dA).(dT)10, poly(dT).(dA)10, poly(dC).(dG)10 and poly(dI).(dC)10. We show that the Klenow fragment is usually much less processive than Pol I and when this is the case it has a much lower exo/pol ratio. At equal processivity, the exo/pol ratios are nearly equal. Furthermore, many factors that influence processivity length (e.g. manganese versus magnesium, inorganic pyrophosphate, ionic strength) influence the exo/pol ratio in the same direction. The study of deaminated poly(dC) replication, where we followed incorporation and excision of both G and A residues, allowed us to assign the origin of the dNMP variations to changes in the 3' to 5' proof-reading activity of Pol I. Similarly, the lower dNMP turnover of the Klenow fragment observed with deaminated poly(dC) was specifically assigned to a decreased 3' to 5' exonuclease activity. The exo/pol ratio generally increased with initiator and decreased with enzyme concentration, in agreement with the model, except for poly(dI).oligo(dC), where it decreased with initiator concentration. However, by terminating chain elongation with dideoxy CTP, we showed directly that, even in this system, excision is relatively inefficient at the beginning of synthesis. Interaction of Pol I with poly(dA).(dT) or with poly(dC).(dG) modifies its exo/pol characteristics in the replication of poly(dI).(dC) and poly(dA).(dT), respectively. The Klenow enzyme is not sensitive to such influences and this correlates with its reduced processivity on the influencing templates. Our results reveal the existence of differences between Pol I and its Klenow fragment that are more profound than has been thought previously.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Base Composition; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA Polymerase I; DNA Replication; Escherichia coli; Kinetics; Models, Biological; Poly dA-dT; Templates, Genetic; Thymine Nucleotides

In a search for the carcinogenic metabolite of diethylstilbestrol, the interactions of 4',4"-diethylstilbestrol quinone with peptides and nucleic acids were investigated. Nonextractable binding of 4',4"-diethylstilbestrol quinone to calf thymus DNA or poly G were observed. However, adduct nucleosides could not be isolated subsequent to enzymatic digestion of nucleic acids. Binding to dGMP or dAMP also occurred, but the initially bound stilbene estrogen could mostly be extracted with 18 extractions using various organic solvents. Non-covalent interactions of 4',4"-diethylstilbestrol quinone with calf thymus DNA were observed spectrally only after exhaustive dialysis of the DNA versus water, but not with native DNA. In chemical reactions of 4',4"-diethylstilbestrol quinone and nucleosides, nucleotides, and amines such as n-pentyl amine, only Z,Z-dienestrol could be identified as reaction product. The quinone did react with mercaptoethanol via Michael addition to the unsaturated carbonyl system to form a stable adduct, 4-(2-hydroxyethylthio)-3,4-di(p-hydroxyphenyl)-2-hexene. It also reacted covalently with sulfur-containing peptides such as reduced glutathione or bovine serum albumin. Partially purified rat liver cytochrome P-450 reductase reduced 4',4"-diethylstilbestrol quinone to E- and Z-diethylstilbestrol. It is proposed that 4',4"-diethylstilbestrol quinone forms unstable adduct intermediates with DNA which decompose with time. Also, covalent binding of 4',4"-diethylstilbestrol quinone to important proteins via thioether linkages may play a role in carcinogenesis.

Topics: Amines; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Dienestrol; Diethylstilbestrol; DNA; Glutathione; Mercaptoethanol; Poly G; Serum Albumin, Bovine