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At least 1,693 records · Page 94Linked to original sources
PubMed1989

Iron ion-dependent modification of bases in DNA by the superoxide radical-generating system hypoxanthine/xanthine oxidase.

Damage to the bases in DNA produced by the hypoxanthine/xanthine oxidase system in the presence of iron ions was studied. The base products in DNA were measured using gas chromatography-mass spectrometry with selected ion monitoring after acidic hydrolysis of DNA and trimethylsilylation. Products identified were cytosine glycol, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These are typical hydroxyl radical-induced products of the bases in DNA. 2,6-Diamino-4-hydroxy-5-formamidopyrimidine was the major product, followed by 8-hydroxyguanine, in DNA treated with hypoxanthine/xanthine oxidase/Fe3+-EDTA. The use of Fe3+ did not cause as much damage to the bases in DNA as did the use of Fe3+-EDTA. In both systems, the formation of the products was inhibited by superoxide dismutase, catalase, dimethyl sulfoxide, mannitol, and desferrioxamine, but inhibitions were much stronger in the systems containing EDTA. Hence formation of hydroxyl radicals by a superoxide radical-assisted Fenton reaction is proposed to account for the results obtained. 2,6-Diamino-4-hydroxy-5-formamidopyrimidine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyguanine were proposed as the products in DNA to measure if one aims to measure DNA products as indices of oxidative DNA damage involving hydroxyl radicals in vivo.

Animals↗
PubMed1987

[Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. X. Hydrolysis of substrates with structural abnormalities].

Interaction of the EcoRII restriction endonuclease with a set of 30-membered substrates having structural anomalies in the recognition site (decreases CCT/AGG) and in adjacent sequences has been studied. A nick in the centre of the EcoRII recognition site between dC and dA residues slows down hydrolysis of the nonmodified strand, whereas the modified one is not cleaved. Removal of the phosphate group from the nick in this substrate does not alter the rate of the cleavage. The absence of one of the phosphate groups in the flanking sequence at a two-base-pair "distance" from the recognition site slows down the enzymatic hydrolysis. Removal of dA or dT out of the EcoRII recognition site blocks the enzymatic reaction. It appears that EcoRII does not interact with the phosphate group between dC and dA residues in the recognition site. Suggestions are made concerning possible contacts of the EcoRII restriction endonuclease with dA- and dT-residues of the recognition site and with the sugar-phosphate backbone of the adjacent nucleotide sequences.

Base Sequence↗
PubMedDate not supplied

[Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks].

A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones.

Autoradiography↗
PubMed1987

[The structure of products of modification of nucleotides and DNA by ethyleneimine and thio-TEPA].

Previously undescribed products of dGMP, GMP, AMP, dCMP and TMP aminoethylation by ethylenimine and N,N',N"-triethylenethiophosphoamide (thio-TEPA) have been obtained and shown to be aminoethyl esters of nucleotides with the free or substituted amino group. In case of dGMP and GMP ethylenimine and tio-TEPA alkylate not only phosphate but also the base residue at the N7 position. The 7-aminoethyl derivatives of dGMP and GMP, which thio-TEPA afforded, were characterized whereas the corresponding ethylenimine derivatives are decomposed under alkaline conditions in the course of the isolation. Possible reasons of extreme instability of these compounds are given. For the first time the ability of thio-TEPA to alkylate DNA at position 7 of guanine residue is shown by means of the luninescence method.

Alkylation↗
PubMed1987

[Modification of esophageal epithelium DNA in the human fetus by N-nitrosomethylbenzylamine (NMBzA)].

Human fetal esophageal epithelium, after being exposed to NMBzA, was found to contain O6-methyldeoxyguanine (O6-MedG), a NMBzA-modified DNA adduct, in tissue DNA by radioimmunoassay and monoclonal antibody, which is highly specific to O6-MedG. The highest level of O6-MedG was 58.83 pMol/mg DNA after adding 5.0 mM NMBzA in vitro. The level of O6-MedG and the concentration of NMBzA followed the dose-effect relationship. O6-MedG could be eliminated from DNA by normal human fetal esophageal epithelium. About 50% of O6-MedG was cleared away in the first 1-2 hours during the post-treatment incubation, which was followed by a slower phase of elimination with 18% left in 24 hours. The results indicate that the human fetal esophageal epithelium can metabolically activate NMBzA in vitro and form O6-MedG, which, as well known, can cause mutagenesis and carcinogenesis and, hence, may most likely be related to the development of human esophageal cancer.

Carcinogens↗
PubMed1986

Radiation-like modification of bases in DNA exposed to tumor promoter-activated polymorphonuclear leukocytes.

Oxygen species generated by human polymorphonuclear leukocytes (PMNs) activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) caused the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdUrd), and (+) and (-) diastereoisomers of cis-thymidine glycol (dTG) in DNA that was exposed to them. There were 9 HMdUrds and 31 dTGs formed per 1 X 10(6) thymidine residues. When Fe(II)/ethylenediaminetetraacetic acid was added to TPA-activated PMNs at 0, 10, 15, and 20 min after TPA, HMdUrd formation increased 5-, 13-, 30-, and 35-fold. Although dTG was initially formed in larger amounts than HMdUrd, it eventually decreased but was still 5-, 6-, 5.5-, and 3-5-fold, respectively, higher than in the absence of iron. From 65 to 1800 times more HMdUrd was formed in DNA when autologous plasma was present during incubation of DNA with TPA-activated PMNs than in its absence. The levels of dTG also varied from about the same as HMdUrd to the nondetectable. Reconstituted human serum transferrin used instead of plasma or Fe(II) also supported the formation of HMdUrd and dTG. When DNA was treated with Fe(II)-reduced H2O2 in the absence of PMNs and TPA, both derivatives were formed. However, the same treatment of marker dTG of dTG-containing polydeoxyadenylic-thymidylic acid caused the decomposition of dTG. Thus, the reduction of hydrogen peroxide by Fe(II) complexed to either ethylenediaminetetraacetic acid or amino acids amy be responsible for the formation of HMdUrd and dTG and for subsequent decomposition of dTG in DNA exposed to the TPA-activated PMNs.

DNA↗
PubMedDate not supplied

[Physical mapping of Streptomyces coelicolor A3(2). V. Structural modifications of actinophage phiC43 DNA molecules].

As shown by genetical and physical methods, the total preparation of phiC43 phage obtained after spontaneous induction of the prophage from S. lividans 803 strain is a heterogenous population. The wild-type phage (phi C43 wt) is only represented in 5--10% of the population. The majority of phage variants are not able to establish the lysogenic state. The structure of DNA molecules of some phages from the total preparations was characterized by electron microscopy of DNA heteroduplexes. Molecules of phiC43 wt DNA appeared to be completely homologous to those of recently studied phiC62 phage, except for two small regions of approximately 0.3 kb in the central part. Phage variants defective in establishment of the lysogenic state were distributed to two groups. One of them consists of deletion variants, the other--deletion/insertion variants. Deletions in DNA molecule of all nonlysogenizing phage overlap. The region of overlapping seems to be responsible for establishment of the lysogenic state. In the same region, deletion of DNA molecules of mutant phiC311 yg2 has been located. Three deletion/insertion variants contain homologous foreign sequences of various length. It is likely that these insertions are fragments of the host chromosomal DNA.

Bacteriophages↗
PubMed1984

Sequence-specific BamHI methylase. Purification and characterization.

BamHI methylase has been purified to apparent homogeneity. The isolated form of the enzyme is a single polypeptide with a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Unlike BamHI endonuclease, which is isolated as a dimer and higher aggregates, the methylase has no apparent higher form. The methylase requires S-adenosyl-L-methionine as the methyl-group donor and is inhibited by Mg2+. The enzyme is also inhibited by 2,3-butanedione and reagents specific for sulfhydryl groups, such as N-ethylmaleimide, which suggests a role for arginine and cysteine residues, respectively. DNA efficiently protects the enzyme against the butanedione modification while S-adenosylmethionine has no effect. In contrast, S-adenosylmethionine protects against cysteine modification while DNA produces only small amounts of protection. Studies on the mechanism of methylation indicate that both strands of the recognition sequence are modified in a single binding event. The sequence specificity of the methylase is relaxed upon the addition of glycerol in the reaction mixture. In the presence of 30% glycerol the enzyme methylates sequences that are also recognized by BamHI endonuclease when acting under conditions of relaxed specificity.

Bacillus↗
PubMed1993

Minute virus of mice-induced modification of the murine DNA polymerase alpha-primase complex permits the salt-induced dissociation of 12S DNA primase and 10S DNA polymerase alpha components.

DNA polymerase alpha-primase complexes in extracts of MVM-infected murine cells were dissociated in the presence of 0.3M KCl to generate a 12S DNA primase and a 10S DNA polymerase alpha that were readily separated by sedimentation in glycerol gradients. A 12S DNA polymerase alpha-primase complex refractory to dissociation in 0.3M KCl was identified in extracts of MVM-infected HeLa cells. In extracts of mock-infected murine and HeLa cells DNA primase and DNA polymerase alpha were not dissociated from each other in 0.3M KCl but remained in a stable complex that sedimented at 10S. We propose that a novel 12S DNA polymerase alpha-primase complex prone to disruption by salt is induced by MVM infection and that the DNA primase component of the complex is modified.

Animals↗
PubMedDate not supplied

[Effect of water on the structural modification and stabilization of DNA from Clostridium perfringens].

By the methods of infrared spectroscopy, piezogravimetry and microcalorimetry the process of forming of the structure and hydration environment of DNA from Clostridium perfringens in the wet films has been investigated. It were found three intervals of relative humidity (r.h.) in which three structural states of DNA: an unordered state (0-44% r.h.), the A-form (46-75% r.h.) and the B-form (80-86% r.h.) are realized. We measured the heat of water evaporation from the DNA-water samples with the differential scanning microcalorimeter. From these calorimetric data the distribution of the water molecules on the bond energy with DNA was determined. The contribution of the water molecules of hydration shell (73%) and hydrogen bonds in the Watson-Crick pairs (27%) to the total energy of stabilization of the helical B-form DNA from Cl. perfringens has been evaluated.

Clostridium perfringens↗
PubMed1998

DNA fragmentation, DNA-protein crosslinks, postlabeled nucleotidic modifications, and 8-hydroxy-2'-deoxyguanosine in the lung but not in the liver of rats receiving intratracheal instillations of chromium(VI). Chemoprevention by oral N-acetylcysteine.

An in vivo study was carried out with the objectives of evaluating (a) the localization of DNA lesions resulting from exposure to chromium(VI) by the respiratory route, (b) the molecular nature of DNA alterations, and (c) modulation of DNA damage by a known chemopreventive agent. To this purpose, Sprague-Dawley rats received intratracheal instillations of sodium dichromate (0.25 mg/kg body weight) for three consecutive days, and the day after the last treatment lung and liver were removed for DNA purification. The results showed a selective localization of DNA lesions in the lung but not in the liver, which can be ascribed to toxicokinetics and metabolic characteristics of chromium(VI). DNA alterations included DNA-protein crosslinks, DNA fragmentation, nucleotidic modifications, and 8-hydroxy-2'-deoxyguanosine. The last two endpoints were evaluated, for the first time in chromium toxicology, by means of postlabeling procedures. This methodology was adapted to the detection of the DNA damage produced by those reactive oxygen species which result from the intracellular reduction of chromium(VI). The oral administration of the thiol N-acetylcysteine completely prevented any induction of DNA lesions in lung cells.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Detection and characterization by 32P-postlabelling of DNA adducts induced by a Fenton-type oxygen radical-generating system.

Reactive oxygen species can give rise to numerous modifications of DNA. We have investigated the formation of such modifications using the nuclease P1 digestion method of the 32P-postlabelling procedure for the detection of DNA damage. Analysis of DNA that had been treated with a Fenton-type system of copper (or iron) ions and H2O2 resulted in the detection of up to ten discrete 32P-labelled spots, displaying chromatographic characteristics similar to aromatic adducts, on PEI-cellulose TLC. Maximum total levels equivalent to 28 adducts/10(8) nucleotides were achieved after 15 min of treatment with Cu2+/H2O2. The formation of adducts was 1.5 times greater if single-stranded rather than double-stranded DNA was employed, suggesting an intrastrand effect. Experiments with 3'-deoxyribonucleotides demonstrated that the adducts detected did not represent base modifications such as 8-hydroxydeoxyguanosine or thymidine glycols. However, treatment of specific dinucleotides (dApdG and dApdA) was found to produce two major adducts that were chromatographically identical by TLC and HPLC to the two major adducts formed in DNA. It is proposed that these species with aromatic adduct-like characteristics are the result of the intrastrand linking of specific adjacent bases in DNA.

Animals↗
PubMed1988

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.

Buffers↗
PubMedDate not supplied

Oxidative DNA damage and cardiovascular disease.

Reactive oxygen species can directly cause covalent modifications to DNA. Alternatively, they can initiate the formation of lipid hydroperoxides, which undergo homolytic decomposition to the alpha,beta-unsaturated aldehyde genotoxins, 4-oxo-2-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroxy-2-nonenal through two quite separate pathways. One pathway involves a complex rearrangement of the alkoxy radical derived from the lipid hydroperoxide. The other pathway involves the intermediate formation of 4-hydroperoxy-2-nonenal. Lipid hydroperoxides can also be derived from the action of lipoxygenases and cyclooxygenases on polyunsaturated fatty acids. 4,5-Epoxy-2(E)-decenal forms etheno-2'-deoxyadenosine adduct with DNA, a mutagenic lesion observed in human tissue DNA samples. Several new ethano- and etheno-DNA adducts have been identified from the reaction of 4-oxo-2-nonenal with DNA. Malondialdehyde, another genotoxic bifunctional electrophile, forms a propano adduct with 2'-deoxyguanosine (M1G-dR) rather than an etheno adduct. Very little is known about the consequences of lipid hydroperoxide-mediated DNA damage in cardiovascular diseases. This should prove to be an important area for future research.

Animals↗
PubMed1999

DNA interactions of cisplatin tethered to the DNA minor groove binder distamycin.

Modifications of natural DNA in a cell-free medium using cisplatin tethered to the AT-specific, minor groove binder distamycin, were studied using various methods of biochemical analysis or molecular biophysics. These methods include: binding studies using differential pulse polarography and flameless atomic absorption spectrophotometry, mapping DNA adducts using a transcription assay, use of ethidium bromide as a fluorescent probe for DNA adducts of platinum, measurement of DNA unwinding by gel electrophoresis, measurement of CD spectra, an interstrand cross-linking assay using gel electrophoresis under denaturing conditions, measurement of melting curves with the aid of absorption spectrophotometry and the use of terbium ions as a fluorescent probe for distorted base pairs in DNA. The results indicate that attachment of distamycin to cisplatin changes several features of the DNA-binding mode of the parent platinum drug. Major differences comprise different conformational alterations in DNA and a considerably higher efficiency of the conjugated drug to form in DNA interstrand cross-links. Cisplatin tethered to distamycin, however, coordinates to DNA with similar base sequence preferences as the untargeted platinum drug. The results point to a unique profile of DNA binding for cisplatin-distamycin conjugates, suggesting that tethering cisplatin to minor groove oligopeptide binders may also lead to an altered biological activity profile.

Antineoplastic Agents↗
PubMed1998

Glutathione-dependent detoxification of alpha-oxoaldehydes by the glyoxalase system: involvement in disease mechanisms and antiproliferative activity of glyoxalase I inhibitors.

The glyoxalase system is a metabolic pathway that catalyses the detoxification of alpha-oxoaldehydes RCOCHO to corresponding aldonic acids RCH(OH)CO2H. It thereby protects cells from alpha-oxoaldehyde-mediated formation of advanced glycation endproducts (AGEs). It is comprised of two enzymes, glyoxalase I and glyoxalase II, and a catalytic amount of reduced glutathione (GSH) as cofactor. It is present in the cytosol of cells of mammals and most micro-organisms. Physiological substrates of the glyoxalase system are: glyoxal--formed from lipid peroxidation and glycation reactions, methylglyoxal--formed from triosephosphates, ketone body metabolism and threonine catabolism, and 4,5-dioxovalerate--formed from 5-aminolevulinate and alpha-ketoglutarate. alpha-Oxoaldehydes react with guanyl residues in DNA and RNA, and with cysteine, lysine and arginine residues in proteins. The modification of DNA induces mutagenesis and apoptosis. The modification of proteins leads to protein degradation and activation of a cytokine-mediated immune response.

Aldehydes↗