Guanine Moiety Research Articles

DNA alkylation by 4,5-dioxovaleric acid, the final oxidation product of 5-aminolevulinic acid.

The heme precursor 5-aminolevulinic acid (ALA) accumulates under pathological conditions, namely, acute intermittent porphyria (AIP) and tyrosinosis, two diseases that are associated with increased liver cancer incidence. This has been previously linked to an enhanced production of reactive oxygen species generated by a metal-catalyzed ALA oxidation process, which was shown to cause DNA single-strand breaks and guanine oxidation within both isolated and cellular DNA. In the present work, we established that the final oxidation product of ALA, 4,5-dioxovaleric acid (DOVA), is an efficient alkylating agent of the guanine moieties within both nucleoside and isolated DNA. Adducts were produced through the formation of a Schiff base involving the N2-amino group of 2'-deoxyguanosine (dGuo) and the ketone function of DOVA, respectively. The modified dGuo nucleosides were characterized, following reduction into stable secondary amines, by extensive NMR, infrared, and mass spectrometry analyses. A method, based on the use of HPLC with electrochemical detection, was then developed for the sensitive measurement of the DOVA-dGuo adducts. Using this assay, we showed that the guanine moieties of isolated DNA can undergo the same reaction as the free nucleoside. The present data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that AIP may be involved in the induction of primary liver cell carcinoma.

Formation of 8-Hydroxyguanine and 2,6-Diamino- 4-hydroxy-5-formamidopyrimidine in DNA by Riboflavin Mediated Photosensitization

Calf thymus DNA was photoirradiated in the presence of riboflavin. Altered bases were detected and quantified by the GC/MS-SIM method after hydrolysis and derivatization of DNA. Seven types of modified purine bases were detected in control DNA. Among them, the yields of 8-OH-Gua and FapyGua increased significantly in DNA photoirradiated with riboflavin, whereas the yields of xanthine, 8-OH-Ade, 2-OH-Ade, FapyAde and hypoxanthine were not affected. A dose dependent increase in the formation of 8-OH-Gua was observed with increasing riboflavin concentration for 30 min irradiation. On the other hand, FapyGua reached plateau at 10 μg/ml of riboflavin for 30 min irradiation. Our results indicate that guanine moiety in DNA is the most susceptible to riboflavin mediated photosensitization.

32P-Postlabeling Detection of DNA Base Adducts in Mice Induced by 4-(Hydroxymethyl)bellzenediazonium Salt, a Carcinogen in Mushroom Agaricus bisporus.

4-(Hydroxymethyl) benzenediazonium salt (HMBD), an ingredient in the edible mushroom Agaricus bisporus, is carcinogenic to mice and mutagenic to bacteria. Formation of carbon-centered radical from HMBD which induces DNA single strand breaks and modifies guanine and adenine moieties of DNA into 8-[4-(hydroxymethyl) phenyl] guanine and 8-[4-(hydroxy methyl) phenyl] adenine has been shown. In the present study HMBD was intraperitoneally administered to mice and in vivo formation of DNA base adducts in liver DNA was investigated by 32P-postlabeling assay. A radioactive spot caused by the modified bases was observed in liver DNA 24 h after the administration which disappeared after 48 h. The base adduct formation may play a role in carcinogenesis of HMBD in mice.

Interaction of HIV Tat Peptides With tRNAPhe from Yeast

We present data on the interaction of arginine-rich peptides of human immunodeficiency virus (HIV-Tat) with tRNAPhe of Saccharomyces cerevisiae. We have found that tRNA forms complexes with the Tat1 peptide of amino acid sequence GRKKRRQRRRA and its mutants where R is replaced by D-arginine, citrulline or ornithine. The structure of tRNA-Tat1 complex was probed by specific RNases digestions and Pb2+-induced cleavage of phosphodiester bond of guanosine. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe binds to Tat peptide and therefore is specifically protected against RNases and is not hydrolyzed by Pb2+ ion. It seems that the peptide-RNA complex formation depends on direct recognition of guanine moieties of tRNA with arginine residues. These interactions are similar to those observed in many DNA-protein complexes, but are different from those previously observed for TAR RNA-Tat complexes.

Stereospecific interactions of chiral cobalt(III)-amine complexes with guanosine 5′-monophosphate

The interactions of two chiral cobalt(III)-amine complex cations, [Co(sep)] 3+ where sep = sepulchrate (1, 3, 6, 8,10,13,16,19-octaazabicyclo[6.6.6]eicosane), and [Co(tetren)(OH)] 2+ where tetren = tetraethylene-pentaamine (1,4,7,10,13-pentaazatridecane), with guanosine 5′-monophospbate (5′-GMP) were characterized in the solid state and in aqueous solution. 1H nuclear magnetic resonance (AMR,) studies show that interactions persist even in dilute solutions of this compound. The 1H NMR spectrum of the products formed when racemic [Co(sep)] 3+ reacts with β- d-5′-GMP reveals a splitting o the AB multiplet of the methylene bridge protons of [CO(sep)] 3+ into two AB multiplets of equal intensity. This is shown to be the result of the formation of the diastereomers [Λ-(+)-Co(sep)] 2(β- d-GMP) 3 and [δ-(−)-Co(sep)] 2(β- d-5′-GMP) 3. The phosphate is not involved in covalent bonding, but could be participating in hydrogen bonding. Fourier transform infrared data of solid [Co(sep)] 2(5′-GMP) 3 · 12H 20 suggest the possibility of hydrogen bonding of N-H sites on the cobalt ligands with N7 and 06 of the guanine base. The compound {[Co(tetren)] 2(5′-GMP)}(ClO 4) 4 · 6H 20 was synthesized and it was found that the phosphate was covalently bonded to the Co(III). The solid decomposes into [Co(tetren) (5′-GMP)] + in aqueous solution. Di-astereomer formation is observed in the 31P NMR when [Co(tetren)(5′-GMP)] + is formed in solution from racemic [Co(tetren)(OH)] 2+ and β- d-5′-GMP 2−, and 1H NMR reveals interactions between the etbylenic protons and guanine moiety. The K′ assoc for [Co(tetren (5′-GMP)] + is 0.17 ± 0.05. Molecular modeling was done and the structures determined correlated well with the experimental data. These Co(III)-amine complex ions illustrate the variety of highly specific interactions which can occur with biomolecules.

Monophotonic ionization of poly[A,G] and DNA in alkaline aqueous solution by 248 nm laser light: Formation of guanyl radical

Photoionization of 2′-deoxyguanosine 5′-monophosphoric acid (dGMP), polyadenylic-guanylic acid (poly[A,G]) and single-stranded DNA (ssDNA) in alkaline aqueous solution by 248 nm laser pulses was investigated. Unlike the reactions in neutral solution, the ionization at pH > 9 was found to be monophotonic. The transition from mono- to biphotonic ionization with increasing pH was shown to be associated with deprotonation of guanine and the corresponding decrease of ionization potential. In the case of Poly[A,G] and ssDNA, photoionization occurs at deprotonated guanine moieties, thus giving guanyl radicals. The quantum yields of formation of hydrated electrons ( Φ e−) were determined at room temperature with KI solutions used as a reference.

Laser-induced modification of DNA and Poly[A,G] at guanine moiety using acetone as photosensitizer

The interactions of triplet acetone with polyadenylic acid (Poly[A]), polyguanylic acid (Poly[G]), polyadenylic-guanylic acid (Poly[A,G]) and single-stranded DNA (ssDNA) were investigated in neutral aqueous solution using KrF (248 nm) laser flash photolysis. The transient absorption spectra and kinetics of DNA and polynucleotides obtained under acetone sensitization demonstrated that the predominant transient species was guanine radical. These novel findings have offered time-resolved evidence for photochemical modification of DNA and Poly[A,G] at guanine moiety.

Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.

Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and Pllb starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and Pllb, and the carboxy terminal proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.

Photophysics of cationic 5,10,15,20-tetrakis-(4- N-methylpyridyl) porphyrin bound to DNA, [poly(dA-dT)] 2 and [poly(dG-dC)] 2: on a possible charge transfer process between guanine and porphyrin in its excited singlet state

The photophysical parameters (fluorescence quantum yield and lifetime, triplet state formation and lifetime) of a cationic prophyrin, i.e. 5,10,15,20-tetrakis(4- N-methylpyridyl) porphyrin (H 2(TMpy-P4)), were determined directly in phosphate buffer solution in the native state and in complexes with calf thymus DNA, [poly(dA-dT)] 2 and [poly(dG-dC)] 2. In [poly(dG-dC)] 2-porphyrin complexes (mostly intercalative), interaction induced an efficient fluorescence quenching and decrease in the triplet state quantum yield. No such effects were observed in [poly(dA-dT)] 2-pophyrin complexes (mostly non-intercalative). The case of DNA was intermediate between the other two. On the basis of the redox properties of the various nucleic bases, it was shown that these observations can be interpreted in terms of fast reversible intermolecular electron transfer from the guanine moiety to the porphyrin in the excited state, when the porphyrin is intercalated with an appropriate geometry within the DNA duplex. These observations and interpretations support a type I (electron transfer) mechanism as the primary event leading to the photodynamic activity of cationic free base porphyrins used as photosensitizers in the process of photoinduced DNA strand breaks.

Influence of the local helical conformation on the guanine modifications generated from one-electron DNA oxidation.

Two major products, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentafuranosyl)amino]-5-(2H )-oxazolone and its imidazole derivative have been generated from one-electron oxidation of the free 2'-deoxyguanosine. The formation of 7,8-dihydro-8-oxoguanine (8-oxodG), not detected in this case, has been observed from DNA exposed to oxidizing agents. Since these compounds are thought to reflect, respectively, either deprotonation or hydration of the transient guanyl radical cation, these findings suggested that the helical structure could influence the chemical decomposition pathway of the guanine moiety. In the present study, we have photoionized DNA sequences by exposure to high-intensity UV (266 nm) laser pulses. Homo- or heteroduplexes, including guanines in various environments as well as Gn runs, were used as templates. Lesions were analyzed, at the nucleotide level, by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. Variations were observed in the relative yield of each type of lesion at individual guanines of the DNA sequences. We found that the Fpg lesions predominate in regions of stable double helix but are decreased in favor of the piperidine ones in regions of destabilization of the helix. Results are discussed in terms of a relationship between intramolecular rearrangements of the guanyl radical cation and the DNA helical conformation and dynamics.

Modelling steric effects in DNA-binding platinum(II)-am(m)ine complexes.

A repulsive energy strategy has been employed in an attempt to delineate the steric contribution to the biological profile of a variety of platinum-am(m)ine complexes. Thus, relative steric descriptors have been calculated for the amine ligands themselves by the Ligand Repulsive Energy (LRE) methodology. This has been extended to a Complex Repulsive Energy (CRE) strategy whereby the steric requirements of the approach of a metal complex to a site on a target molecule may be evaluated. Specifically, the monodentate approach of a variety of platinum-am(m)ine complexes to the N7 site of a guanine moiety has been considered. The steric descriptors thus obtained have been used in QSAR analysis, resulting in improved regression equations. Attempts have also been made to relate the above descriptors to various biological indicators for given series of complexes. These investigations suggest an optimum steric requirement for minimum toxicity, which could aid in the rational design of such agents.

Copper(II) interactions with an experimental antiviral agent, 1-deoxynojirimycin, and oxygen activation by resulting complexes

1-deoxynojirimycin (DNJ), a 5-imino analog of 1-deoxyglucose, is a potent inhibitor of α-glucosidase I. DNJ and its derivatives have been considered as experimental drugs against human HIV-1 and hepatitis B viruses. Since amino and imino ligands have a high affinity for copper, it seems possible that biological activity of DNJ may be, at least in part, modulated by tissue copper. To test this possibility, potentiometric and spectroscopic studies of the complexation of DNJ by cupric ions were performed in order to obtain thermodynamic and structural background for further pharmacologic investigations. The effect of histidine, a major tissue copper carrier, on coordination equilibria was also studied. Results indicate that DNJ and Cu(II) form two stable complexes at physiological pH, CuH −1(DNJ) 2 + and CuH −2(DNJ) 2, involving Cu(II) chelation by the N-5 and O-6 donor atoms. In the presence of histidine, ternary complexes are also formed, of which the CuDNJHis + species is stable in the physiological pH range. Binary Cu(II)-DNJ complexes are extremely effective mediators of in vitro oxidation of the guanine moiety in both 2′-deoxyguanosine (dG) and DNA to 8-oxoguanine (8-oxo-dG) and of DNA double strand scission by ambient O 2 or H 2O 2. This mediation is suppressed by histidine in dG, but not in DNA. The results suggest that tissue Cu(II) may greatly enhance nonspecific cytotoxic effects of systemically administered DNJ through oxidative damage mechanisms, and therefore the prospective use of DNJ for therapeutic purposes must be developed with caution. On the other hand, however, the expected high genotoxic potential of synthetic Cu(II)-DNJ complexes may be used against viruses by means of targeted delivery of these complexes to the infected cells.

Homo-N-nucleosides: Incorporation into oligonucleotides and antiviral activity

Homo-N-nucleosides can be efficiently synthesized from 2-deoxyribose. When incorporated in an oligonucleotide, the compounds have a detrimental influence on duplex stability and on the catalytic activity of hammerhead ribozymes. However, homo-N-nucleosides with a guanine or adenine base moiety do exhibit selective antiviral activity against herpes simplex virus (HSV-1 and HSV-2).

Trace measurements of nucleic acids using flow injection amperometry

Flow injection analysis with amperometric detection is used for quantitating nucleic acids down to picogram level. The procedure relies on the oxidation of the bound guanine moiety at the untreated carbon paste detector, and does not require any prior derivatization or hydrolysis reactions. The response is evaluated with respect to flow rate, operating potential, type and concentration of the nucleic acid, and other variables. At a fixed potential of +1.0 V ( vs. Ag AgCl ) the detection limits for double-stranded DNA, single-stranded DNA and tRNA are 460, 540 and 750 pg, respectively. Good reproducibility (R.S.D.s of 1.9–2.9%) is observed for prolonged series ( n = 30). The attractive behavior of the carbon paste detector holds great promise for monitoring nucleic acids in Chromatographic effluents, and should facilitate new applications in molecular biology.

Studies on the mechanisms of haloacentronitrile-induced genotoxicity IV: In vitro interaction of haloacetonitriles with DNA

Haloacetonitriles, which are water chlorination by-products, are mutagens, carcinogens and teratogens. In vitro, the reaction of the haloacetonitriles bromoacetonitrile (BAN), chloroacetonitrile (CAN), dichloroacetonitrile (DCAN) and trichloroacetonitrile (TCAN) with calf thymus DNA produced fluorescent DNA derivatives. The reactivity of haloacetonitriles towards DNA was in the order of BAN > CAN > DCAN > TCAN. The emission fluorescence spectra of the reaction product(s) of various haloacetonitriles with DNA has a peak at 404 nm at fixed excitation wavelength (300 nm). The fluorescence intensity of the reaction product(s) was pH dependent with an optimum intensity at pH 7.4. The DNA interaction was dependent on haloacetonitrile concentration. Higher affinity of haloacetonitriles towards single-stranded DNA (SS-CT-DNA) than towards double-stranded DNA (DS-CT-DNA) was observed. To characterize the type of fluorescent product(s) formed, samples of the reaction product of SS-CT-DNA with BAN were hydrolysed (a) enzymatically by micrococcal nuclease and spleen phosphodiesterase to nucleotides and nucleotide derivative(s) or (b) chemically by formic acid to nucleobases and nucleobase adducts. The hydrolysates were analysed by reversed phase HPLC. A major fluorescent peak was detected in the enzymatic hydrolysate that was not present in unreacted DNA. In the acid hydrolysate, one fluorescent peak was detected that was not present in unreacted DNA. Authentic 7-(cyanomethyl)guanine was synthesized by the reaction of CAN with 2′-deoxyguanosine and the product was purified and characterized spectroscopically. The product, 7-(cyanomethyl)guanine, was found to be chromatographically and spectroscopically identical to the fluorescence product that was obtained following haloacetonitrile-DNA interaction. This study shows that haloacetonitriles, in vitro, are capable of alkylating DNA at the guanine moiety to form a 7-(cyanomethyl)guanine adduct.

Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) in primary rat hepatocytes.

Cooking of proteinous food results in the formation of heterocyclic amines. Among these, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) has been identified as a mutagenic pyrolysis product of soya protein and has been detected in grilled or pan fried meat. It was subsequently proven to be carcinogenic in mice and, recently, in rats and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound, 2-nitro-3-methyl-9H-pyrido[2,3-b]indole (MeNalphaC), was prepared and shown to be a direct acting mutagen in the Ames Salmonella reversion assay. When MeNalphaC was chemically reduced in the presence of DNA a major DNA adduct was detected using the 32P-postlabelling assay. This major adduct was characterized by UV spectroscopy and mass spectrometry as N-(deoxyguanosin-8-yl)-2-amino-3-methyl-9H-pyrido[2,3-b]indole. This structure was corroborated by identification of the modified base as a guanine moiety modified at the C8 position as judged by chromatographic and spectral comparison with a standard synthesized from acetylated guanine-N3-oxide and MeAalphaC was characterized by UV/Vis and 1H NMR spectroscopy and mass spectrometry. Treatment of primary rat hepatocytes with MeAalphaC (100 microM, 24 h) resulted in adduct levels of 9.8 fmol/microg DNA as determined by 32P-postlabelling analysis. Using HPLC analysis, two major 32P-labelled adducts were observed accounting for 80 and 13% of total binding respectively. The major adduct was chromatographically indistinguishable from the synthetic deoxyguanosine-C8 adduct using both ion-exchange thin layer or reversed-phase HPLC. Although MeAalphaC is formed only in low p.p.b. levels in cooked food, the contribution to the human carcinogenic risk that might be imposed by heterocyclic amines is not to be neglected.

Reaction of singlet oxygen with 2'-deoxyguanosine and DNA. Isolation and characterization of the main oxidation products.

The reaction of singlet molecular oxygen with 2'-deoxyguanosine and DNA was studied. Emphasis was placed on the identification and characterization of the main methylene blue mediated type II (singlet oxygen) oxidation products of 2'-deoxyguanosine and its corresponding 3',5'-di-O-acetylated derivative. Two major oxidation products of 2'-deoxyguanosine were isolated and characterized by mass spectrometry analysis and extensive 1H and 13C NMR measurements as the two 4R* and 4S* diastereomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine. The addition of 1O2 was also found to occur to the base moiety of the corresponding 3',5'-di-O-acetylated derivative. Methylene blue mediated photosensitization of 2'-deoxyguanosine led also to the production of 7,8-dihydro-8-oxo-2'-deoxyguanosine, but in a relatively lower yield with respect to the two above diastereomers. The participation of singlet oxygen in the mechanism of formation of these oxidation products was confirmed. A reasonable mechanism involving the transient formation of an unstable endoperoxide produced through a Diels-Alder 1,4-cycloaddition of singlet oxygen to the purine ring is suggested. Quantitative analysis allowed us to demonstrate that the two diastereomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine are the main singlet oxygen oxidation products of the guanine moiety within nucleosides, whereas 7,8-dihydro-8-oxoguanine was found to be the major 1O2 oxidation product of guanine in double-stranded DNA.

Photoadduct between tris(1,4,5,8-tetraazaphenanthrene)ruthenium(II) and guanosine monophosphate–a model for a new mode of covalent binding of metal complexes to DNA

Visible light irradiation of [Ru(TAP)3]2+(TAP = 1,4,5,8 -tetraazaphenanthrene) in aqueous solutions containing 5′-guanosine-monophosphate (GMP) and subsequent treatment with 1 mol dm–3 HCl yields Ru(TAP)2(2-TAP-G)2+, in which the guanine moiety is bound via N-2 to C-2 of one of the TAP ligands.

Molecular electric field mapping and structure-activity relationships for some human O6-alkylguanine-DNA alkyl transferase depleting agents

Electric field mapping has been carried out for certain O 6- and S 6-substituted purine derivatives, some of which are known to have the ability to deplete the human DNA guanine O 6-alkylation damage repair enzyme called O 6-alkylguanine-DNA alkyltransferase (AGT). Geometries of all the molecules are optimized using the latest semiempirical method, PM3. Point charges at different atomic sites are obtained using Mulliken population analysis. There exists a reasonably good correlation between the average electric field values near the N 7 atom of the guanine moiety in these molecules and the observed AGT depleting activities. It indicates involvement of the N 7 site of the guanine moiety in the reaction of these molecules with AGT, in agreement with earlier suggestions.

Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5‘-triphosphate to xanthosine 5‘-triphosphate.

The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase. Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis. The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP. The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues. The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme. In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate. The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP. In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP. The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme. The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP. These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E. coli is a determinant for GTP-specificity.