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At least 649 records · Page 36Linked to original sources
PubMed1991

Interaction of RSU 1069 and 1137 with DNA in vitro. Biological implications and mechanistic aspects.

We have examined the capacity of the nitroimidazole aziridine antitumour drug RSU 1069 to react with DNA in vitro in order to get a better understanding of its mechanism of action. Moreover, we have utilized biologically active phi X174 DNA to investigate the biological relevance of the chemical DNA modification induced by the drug. Incubation of RSU 1069 in the presence of single-stranded phi X174 DNA resulted in extensive inactivation of the DNA, which is dependent on the concentration of drug and temperature. Only about 2% of the inactivating damage can be attributed to strand breakage. The main damage most probably consists of base damage, of which a part is non-lethal and alkali-labile which in turn can be converted into lethal lesion and subsequently into a break applying a post-incubation alkali treatment. Furthermore, from the dependence of the inactivation and also the formation of breaks on pH and ionic strength, it is concluded that the reaction most probably takes place between a protonated RSU 1069 and a negative DNA coil and that the damage pattern reflects the difference in reactivity of RSU 1069 with the phosphate groups and the bases in DNA. Comparison between RSU 1069 and its ring-open hydrolysis product RSU 1137 revealed that (lethal) damage induced in the DNA must be ascribed to the alkylating properties of the aziridine moiety.

Alkalies↗
PubMed1987

Breakage of double-stranded DNA due to single-stranded nicking.

Enzymes such as pancreatic deoxyribonuclease (DNase I) nick the single strands of double-stranded DNA. Two nicks sufficiently close on opposite strands will lead to breakage of the DNA molecule. This paper gives a mathematical model for the breakage of circular, supercoiled DNA under the action of an enzyme which nicks at random sites (or at preferred sites, these being in abundance and randomly positioned around the circle). After the first nick the DNA loses its supercoiled structure; after many nicks it breaks to become topologically linear; further nicks lead to fragmentation of this linear form. Formulae are given for the proportions of DNA molecules in each of the four classes: supercoiled; nicked but still circular; linear; fragmented. Formulae are also presented for the case when there is, in addition to nicking, simultaneous action of an endonuclease which produces direct double-stranded breaks in the DNA. Finally, a general theory is given for the case where a third type of enzyme, topoisomerase I, is operative, with all three DNA modifications taking place simultaneously.

DNA Topoisomerases, Type I↗
PubMed1996

Characterization of mutations induced by 300 and 320 nm UV radiation in a rat fibroblast cell line.

The cytotoxic and mutagenic activities of monochromatic ultraviolet light (UV) at four wavelengths (254, 290, 300 and 320 nm) were determined using a rat fibroblast cell line CREF stably infected with a retroviral vector carrying the neo and HSV-tk markers. In this system, mutations can be positively detected as acyclovir-resistant colonies. Although the action spectra for these activities closely fit some of the previously reported spectra for photochemical DNA modifications, erythema, cell killing and mouse skin carcinogenesis, they diverge at 320 nm from the absorption spectrum for DNA and the action spectrum for bacterial inactivation and mutagenesis. Structural comparison of the HSV-tk mutants detected after irradiation with 300 and 320 nm UV revealed (1) CC dimers and C oligomers as predominant targets at both wavelengths; (2) increased incidence of relatively large deletions at 300 nm; and (3) greatly increased frequency of tandem double mutations at both wavelengths and of clustered multiple mutations at 320 nm. These results suggest the involvement of distinct mechanisms specifically operating, or becoming evident, in UV-mediated mutagenesis at these different wavelengths in mammalian cells.

Acyclovir↗
PubMed2002

Making AppDNA using T4 DNA ligase.

5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions. Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site. This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases. We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.

Adenosine Monophosphate↗
PubMed1995

Dinucleotide relative abundance extremes: a genomic signature.

Early biochemical experiments established that the set of dinucleotide odds ratios or 'general design' is a remarkably stable property of the DNA of an organism, which is essentially the same in protein-coding DNA, bulk genomic DNA, and in different renaturation rate and density gradient fractions of genomic DNA in many organisms. Analysis of currently available genomic sequence data has extended these earlier results, showing that the general designs of disjoint samples of a genome are substantially more similar to each other than to those of sequences from other organisms and that closely related organisms have similar general designs. From this perspective, the set of dinucleotide odds ratio (relative abundance) values constitute a signature of each DNA genome, which can discriminate between sequences from different organisms. Dinucleotide-odds ratio values appear to reflect not only the chemistry of dinucleotide stacking energies and base-step conformational preferences, but also the species-specific properties of DNA modification, replication and repair mechanisms.

Animals↗
PubMed1982

Mechanism for the loss of preferential benzo [a] pyrene binding to the linker DNA of chromatin.

We have examined the fate of the asymmetric chromosomal distribution of DNA adducts generated by the chemical carcinogen r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). Treatment of mouse embryo cells with BPDE results in 3.5 times more binding to the linker DNA regions between nucleosome cores than to the nucleosome core DNA itself, but 24 h post-treatment incubation of these cells leads to a loss of this non-random binding. A similar result was obtained when post-treatment incubation was carried out in the presence of hydroxyurea indicating that factors other than DNA replication are responsible for this changes in adduct distribution. However in the case of excision repair deficient xeroderma pigmentosum (XP12/BE) cells the non-random adduct distribution was stable over a period of 48 h, whereas with excision repair proficient XP variant (XP4/BE cells, loss of preferential binding did occur. There results indicate that the loss of non-random nucleosomal DNA modification with time can be accounted for by the preferential removal of adducts from micrococcal nuclease sensitive linker DNA and further, demonstrates that in certain cells at least, the relative position of nucleosome core structures on DNA remains unchanged over a period of at least 48 h.

Animals↗
PubMed1997

Photochemical and photobiological studies of a furonaphthopyranone as a benzo-spaced psoralen analog in cell-free and cellular DNA.

Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glycosylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29 +/- 0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D2O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert-butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (Ered = -1.93 +/- 0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (delta GET ca -6 kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures.

Animals↗
PubMed2005

Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs.

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4(+) lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.

5' Flanking Region↗
PubMed1992

Structural modifications of camptothecin and effects on topoisomerase I inhibition.

Camptothecin (1), a potent antitumor alkaloid, is known to inhibit topoisomerase I, an enzyme that relaxes supercoiled DNA. Modifications have been made to the B, D, and E rings of this natural product. Specifically, compounds 2-10 either have an ester moiety in place of the E ring lactone, a methyl ester attached to position 14, a saturated (or nonexistent) deaza B ring, or contain a combination of these permutations. We have conducted in vitro assays against the topoisomerase I relaxation reaction which verify the necessity for a lactone in the E ring. Furthermore, steric requirements at position 14 are shown to be crucial for activity, and planarity of the A and B rings of camptothecin is also implicated in the ability of the drug to inhibit topoisomerase I. Speculation on the nature of the drug binding pocket is presented.

Animals↗
PubMed2004

Oxidative DNA damage and disease: induction, repair and significance.

The generation of reactive oxygen species may be both beneficial to cells, performing a function in inter- and intracellular signalling, and detrimental, modifying cellular biomolecules, accumulation of which has been associated with numerous diseases. Of the molecules subject to oxidative modification, DNA has received the greatest attention, with biomarkers of exposure and effect closest to validation. Despite nearly a quarter of a century of study, and a large number of base- and sugar-derived DNA lesions having been identified, the majority of studies have focussed upon the guanine modification, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OH-dG). For the most part, the biological significance of other lesions has not, as yet, been investigated. In contrast, the description and characterisation of enzyme systems responsible for repairing oxidative DNA base damage is growing rapidly, being the subject of intense study. However, there remain notable gaps in our knowledge of which repair proteins remove which lesions, plus, as more lesions identified, new processes/substrates need to be determined. There are many reports describing elevated levels of oxidatively modified DNA lesions, in various biological matrices, in a plethora of diseases; however, for the majority of these the association could merely be coincidental, and more detailed studies are required. Nevertheless, even based simply upon reports of studies investigating the potential role of 8-OH-dG in disease, the weight of evidence strongly suggests a link between such damage and the pathogenesis of disease. However, exact roles remain to be elucidated.

DNA Adducts↗
PubMed1998

The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of immunocytochemical determination with detection in isolated DNA.

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH3)2d(pGpG) (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis-Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible--at least in part--for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to plantinum-DNA damage may also be involved.

Animals↗
PubMed2006

Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription.

Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thaliana genes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.

Arabidopsis↗
PubMed1995

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.

Bacillus subtilis↗
PubMed1988

DNA binding by [2,5-14C]N-nitrosopyrrolidine in excision-repair proficient and deficient strains of Salmonella. Evidence for a major premutagenic adduct.

Little is known about the nature and possible genotoxic effects of the DNA adducts formed by N-nitrosopyrrolidine (NPYR) in whole animals. DNA binding in DNA isolated from [2,5-14C]NPYR-treated Salmonella was studied and attempts were made to monitor DNA adducts and correlate DNA binding with mutagenesis. NPYR was metabolized by hamster liver S-9 fraction in the presence of S.typhimurium TA1535 (uvrB-) or TA1975(uvrB+). DNA isolated from TA1535 contained about three times as much radioactivity as that isolated from TA1975, and NPYR-induced mutagenesis was several-fold higher in TA1535. The fraction of radioactivity incorporated into TA1535 was approximately 10(-5). Thermal hydrolysis of the 14C-containing DNA at neutral pH, followed by precipitation, released approximately 2/3 of the radioactivity into the supernatant. HPLC analysis of the supernatant revealed one major peak. This peak was absent in DNA from TA1975. Acid hydrolysis of the DNA precipitate after neutral hydrolysis released most of the residual radioactivity. Several small peaks were observed after HPLC analysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate. These results demonstrate that NPYR is capable of binding to Salmonella DNA yielding one major product after hydrolysis and this DNA binding product appears to be repaired by the excision repair system. The fact that the major peak of radioactivity released from Salmonella is only found in the strain which is efficiently reverted by NPYR suggests that mutagenesis is dependent on the DNA modification leading to this peak.

Animals↗
PubMed1997

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.

Adenosine Monophosphate↗
PubMed1994

DNA adduct assay in cervical epithelium.

Numerous epidemiological studies have shown that there is an association between smoking and cervical cancer. However, the essential evidence to show whether this relationship is casual or causal is lacking. The demonstration of DNA modification by tobacco components in the cervical epithelium would provide biochemical evidence to support a causal role. In this study, DNA from 39 cervical biopsies was analysed for the presence of DNA adducts using the 32P-postlabeling technique. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and nonsmokers. DNA samples from smokers [identified from questionnaire] were found to have significantly higher adduct levels than nonsmokers (Mann-Whitney one-tailed U-test, 95% CI > 0.339, P = 0.024). Exclusion of the women whose urinary cotinine levels did not confirm their self-reported smoking status (smoker or nonsmoker) increased this significance (95% CI > 0.508, P = 0.01). Women who had abnormal cervical smears had significantly higher DNA adduct levels than those with normal smears (95% CI > 0.439, P = 0.015). Monitoring of women with high DNA adduct levels may be a way of identifying women at risk of cervical cancer. These findings demonstrate that tobacco smoking by women leads to elevated levels of DNA adducts in cervical epithelium and provides the biochemical evidence to support the concept that smoking is a cause of cervical cancer.

Adult↗
PubMed2004

Acetoaminophen-induced accumulation of 8-oxodeoxyguanosine through reduction of Ogg1 DNA repair enzyme in C6 glioma cells.

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome p450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.

Acetaminophen↗
PubMed1989

Release of chloroethyl ethyl sulfide-modified DNA bases by bacterial 3-methyladenine-DNA glycosylases I and II.

Treatment with chloroethyl ethyl sulfide introduces the following modified bases into DNA: 7-ethylthioethylguanine, 3-ethylthioethyladenine, and O6-ethylthioethylguanine. Using the ethylthioethylated bases as models for DNA modifications involving relatively bulky alkyl groups, we have investigated the release of these bases by Escherichia coli 3-methyladenine-DNA glycosylases I and II. 3-Methyladenine-DNA glycosylase I releases only 3-ethylthioethyladenine from chloroethyl ethyl sulfide-modified DNA, but does so at a rate which exceeds the rate of release of 3-methyladenine (m3A) from methyl nitrosourea-modified DNA under these conditions. 3-Methyladenine-DNA glycosylase II releases both 3-ethylthioethyladenine and 7-ethylthioethylguanine at rates approximating or exceeding the rate of release of m3A from methylnitrosourea-modified DNA. We conclude that these glycosylases may offer some protection against the toxicity of agents which introduce bulky groups into E. coli DNA.

Adenine↗