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PubMed2006

Direct electrochemistry of endonuclease III in the presence and absence of DNA.

The electrochemistry of the base excision repair enzyme Endonuclease III (Endo III) in the presence and absence of DNA has been examined on highly oriented pyrolytic graphite (HOPG). At the surface modified with pyrenated DNA, a reversible signal is observed at 20 mV versus NHE for the [4Fe-4S]3+/2+ couple of Endo III, similar to Au. Without DNA modification, oxidative and reductive signals for the [4Fe-4S] cluster of Endo III are found on bare HOPG, allowing a direct comparison between DNA-bound and free redox potentials. These data indicate a shift of approximately -200 mV in the 3+/2+ couple upon binding of Endo III to DNA. This potential shift reflects a difference in affinity for DNA of more than 3 orders of magnitude between the oxidized 3+ and reduced 2+ protein and provides quantitative support for our model utilizing DNA-mediated charge transport to redistribute base excision repair enzymes in the vicinity of damaged DNA.

DNA↗
PubMed1998

Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer.

Genomic imprinting is the epigenetic mechanism that distinguishes whether the loci that are inherited from the maternal or paternal genome lead to parent-specific gene expression. The mechanism also regulates development in mammalian embryos. Genomic imprinting is established after implantation according to the specific markers that are imposed on the genome during gametogenesis; the allele-specific gene expression is then maintained throughout embryogenesis. The genomic imprinting markers are erased and renewed on an own-sex basis only in cells that differentiate into germline cells. This report shows that the epigenetic modifications that occur during oogenesis perform the crucial function of establishing the allele-specific expression of imprinted genes, and also suggests that the epigenetic DNA modification is related to the reprogramming and aberrant development seen in manipulated embryos.

Animals↗
PubMed1997

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases.

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.

Animals↗
PubMed2002

DNA methylation patterns at relapse in adult acute lymphocytic leukemia.

PURPOSE: Aberrant DNA methylation of promoter-associated CpG islands is an epigenetic DNA modification observed in acute leukemias that in certain cases has been associated with a poor prognosis and increased relapse rates. To study the role of DNA methylation in relapse mechanisms in acute lymphocytic leukemia (ALL), we have compared the methylation status of five genes at the time of initial presentation and at first relapse in 25 adult patients with ALL. EXPERIMENTAL DESIGN: Genes studied included the estrogen receptor (ER), multidrug resistance gene 1 (MDR1), p73, p15, and p16. DNA was extracted from paraffin-embedded bone marrow biopsies. DNA methylation was analyzed using PCR of bisulfite-modified DNA. RESULTS: Results indicate that methylation at the time of relapse was stable in 92% of patients for p73, 88% for ER, 80% for p16, 72% for MDR1, and 60% for p15. Only one case had p16 methylation at initial presentation, whereas 6 patients (P = 0.0001) had methylation at relapse. Three cases had concomitant methylation of p15 and p16 at relapse. The degree of MDR1 methylation inversely correlated with the presence of MDR1 expression as detected by immunohistochemistry. Eighteen patients (72%) had acquired no or one methylation change, whereas the rest (28%) had methylation changes in two or three genes. No clinical-biological correlations were found between methylation of any particular gene or pattern. CONCLUSIONS: In summary, DNA methylation patterns are stable in a majority of patients with relapsed ALL, but a subset of patients acquire new methylation changes, in particular affecting cell cycle regulatory genes.

Adolescent↗
PubMed1987

Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene.

6-Nitrochrysene (NC) and 6-aminochrysene (AC) have been shown to be potent lung and liver carcinogens when administered in multiple i.p. doses to preweanling mice. 1,6-Dinitropyrene has been shown to be a strong hepatocarcinogen but a weak lung carcinogen in this same bioassay. We have examined carcinogen-DNA adduct profiles in the target tissues of preweanling male CD-1 mice following administration of single or multiple doses of these compounds. Depending on the tissue and the dosing schedule, the total level of DNA modification in animals dosed with [3H]NC was 2- to 9-fold higher than in animals dosed with [3H]AC. Regardless of the dosing schedule, DNA isolated from the lungs and livers of both [3H]NC- and [3H]AC-treated preweanling male mice contained a single major and chromatographically identical adduct. This major adduct, which accounted for as much as 90% of the total carcinogen-DNA adducts in enzymatic hydrolysates from treated animals, was chromatographically distinct from the major C8-purine-substituted adducts formed from the reaction of N-hydroxy-AC with calf thymus DNA. In contrast to the results obtained with NC and AC, the major carcinogen-DNA adduct formed in the livers of mice treated with [3H]-1,6-dinitropyrene was found to cochromatograph with 1-N-(deoxyguanosin-8-yl)amino-6-nitropyrene, a product derived from N-hydroxy-1-amino-6-nitropyrene. Since NC and its nitro-reduced derivative, AC, yielded an identical carcinogen-DNA adduct in vivo and this adduct was not derived from N-hydroxy-AC, we conclude that the metabolic activation of NC in the neonatal mouse must involve some previously undescribed combination of ring-oxidation and nitro-reduction pathways. This activation pathway could be an important factor in determining the potency of NC and AC as carcinogens in this bioassay system.

Animals↗
PubMed1977

DNA repair in Bacillus subtilis. I. The presence of an inducible system.

Following UV irradiation of Bacillus subtilis there is a coordinate induction of: 1) a new protein, 2) a W-reactivation system, 3) a DNA modification system, and 4) prophages. These functions are induced following UV irradiation of repair proficient bacteria and mutants deficient in excision repair (UVR-1) and DNA polymerase I activity (polA5). However, they are not induced, or are impaired in their ability to be induced in bacteria containing the recA1 and the recG13 mutations. This inducible system is compared to the SOS system observed in E. coli.

Bacillus subtilis↗
PubMed1999

Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke.

The assessment of pathological effects produced by environmental tobacco smoke in humans is controversial in epidemiological studies. On the other hand, animal models are poorly sensitive to smoke carcinogenicity. We designed an experimental study assessing the tissueselective formation and persistence of DNA adducts in smoke-exposed rats. Sprague-Dawley rats were exposed for 6 h per day, 5 days per week, to environmental smoke resulting from a mixture of sidestream and mainstream smoke generated from Kentucky 2R1 reference cigarettes. The total particulate matter was in the range of 73-93 mg/m(3). DNA adducts were measured by (32)P-post-labelling in rat organs (lung, heart, liver, bladder and testis), tissues (dissected tracheal epithelium) and cells [isolated bronchoalveolar lavage (BAL) cells]. A time-related increase of (32)P-post-labelled DNA modifications was detectable by autoradiography, in the form of massive diagonal radioactive zones and individual spots. Top levels were reached after 4-5 weeks of exposure. The ratio of smoke-induced DNA adducts to the background levels detected in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1. 1 in the liver. Appearance of DNA adducts in the lung was also revealed by synchronous fluorescence spectrophotometry. Smoke-related oxidative damage was demonstrated by a significant enhancement of 8-hydroxy-2'-deoxyguanosine in lung DNA. In parallel, there was a time-related induction of lung microsomal arylhydrocarbon hydroxylase activity, an elevation in cytosolic glutathione S-transferase activity, and a moderate but progressive and significant depletion of reduced glutathione. After discontinuing exposure to environmental cigarette smoke for 1 week, DNA adduct levels significantly dropped in the lung, tracheal epithelium, heart and bladder. The decrease was evident but not statistically significant in BAL cells, and was negligible in the heart. The selective localization and the differential persistence of these promutagenic nucleotide modifications in rat organs, tissues and cells suggest that exposure to environmental cigarette smoke, at least under the high exposure regimens used in experimental studies, may pose a potential risk of developing mutation-related diseases.

8-Hydroxy-2'-Deoxyguanosine↗
PubMedDate not supplied

[Modification of human DNA polymerase alpha by N-acetylimidazole].

The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.

DNA Polymerase II↗
PubMed2003

Effects of X-irradiation in early ontogenesis on the longevity and amount of the S1 nuclease-sensitive DNA sites in adult Drosophila melanogaster.

The long-term consequences of the X-irradiation of Drosophila melanogaster fruit fly one-hour eggs with doses of 0.25, 0.50, 0.75, 1, 2 and 4 Gy were investigated. Longevity hormesis was observed in males exposed to 0.5 Gy and 0.75 Gy, but no longevity increase was observed in females. The electrophoretic analysis has shown that the amount of the DNA segments resulting from cleavage in S1 nuclease-sensitive sites (<3 kb) reached 39.2% of the total DNA from control males. DNA from the irradiated males had a smaller amount of such fragments (10-30% in different experimental groups). These findings indicate that the longevity hormesis may be associated with irradiation-induced long-term structural and/or functional DNA modifications.

Animals↗
PubMed1981

5-Azacytidine induction of mouse endogenous type C virus and suppression of DNA methylation.

5-Azacytidine was found to induce the expression of BALB:virus-1 and BALB:virus-2 from K-BALB cells and ecotropic endogenous virus from AKR2B cells. Efficiency of the induction was high and comparable to that by 5-bromodeoxyuridine. The level of methylcytosine in newly synthesized DNA was drastically decreased when K-BALB cells were treated with 5-azacytidine. There was an inverse relationship between the level of DNA modification and the frequency of virus expression.

Animals↗
PubMed1998

High fat diet induced oxidative DNA damage estimated by 8-oxo-7,8-dihydro-2-deoxyguanosine excretion in rats.

The role of dietary fats and energy in carcinogenesis has been partly related to oxidative damage to DNA. We have investigated the effect of dietary fat content and saturation on the urinary excretion of 8-oxo-7,8dihydro-2'-deoxyguanosine (8-oxodG) in male and female rats. Groups of Fischer F344 rats (n = 6-10) were fed control chow (3.4% fat) or diets containing 21.8% corn oil or 19.8% coconut oil + 2% corn oil for 12-15 weeks. At the end of the diet intervention period 24h urine was collected for determination of 8-oxodG by HPLC. In the male groups fed control, corn oil and coconut oil diet the excretion of 8-oxodG was 403+/-150, 932+/-198 and 954+/-367pmol/kg 24 h, respectively (p < 0.05). In the female groups fed control and corn oil diet the excretion of 8-oxodG was 752+/-80 and 2206+/-282 pmol/kg 24 h, respectively (p < 0.05). Calculated per whole animal the excretion was 137+/-51, 324+/-70 and 328+/-128 pmol/24 h in the control, corn and coconut oil male groups and 156+/-21 and 464+/-56 pmol/24 h in the control and corn oil female groups, respectively ( p < 0.05). Thus, per animal or per consumed energy there was much less difference in 8-oxodG excretion between the corresponding male and female groups and only significant difference between the high fat groups. There was a close correlation (r = 0.7; p < 0.05) between 8-oxodG excretion and the energy intake. The present study suggests that a high fat diet increases oxidative DNA modification substantially irrespective of the saturation level of the fat. Energy intake appears to be the major determinant of the rate of modification.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation.

Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications. Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis. In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells. Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions. The percentage was dependent on the species ultimately responsible for the DNA damage and was approx. 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA.

Animals↗
PubMed2004

Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry.

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.

Cell Line↗
PubMed2005

CpG methylation of the CENP-B box reduces human CENP-B binding.

In eukaryotes, CpG methylation is an epigenetic DNA modification that is important for heterochromatin formation. Centromere protein B (CENP-B) specifically binds to the centromeric 17 base-pair CENP-B box DNA, which contains two CpG dinucleotides. In this study, we tested complex formation by the DNA-binding domain of CENP-B with methylated and unmethylated CENP-B box DNAs, and found that CENP-B preferentially binds to the unmethylated CENP-B box DNA. Competition analyses revealed that the affinity of CENP-B for the CENP-B box DNA is reduced nearly to the level of nonspecific DNA binding by CpG methylation.

Autoantigens↗
PubMed1996

[Increase in the effectiveness of site-specific cleavage of target DNA by tetranucleotide bleomycin derivatives using oligonucleotide-effectors].

The efficiency of site-specific interaction of target DNA with bleomycin derivatives of short nucleotides can be significantly increased using flanking effector oligonucleotides. The cleavage of a 20-mer single-stranded target DNA by a tetranucleotide containing a bleomycin A5 residue at the 5'-end was studied in the presence of effector oligonucleotides bearing phenazine residues at the 5'- and 3'-ends. In the presence of two effectors, the extent of the target DNA modification at 37 degrees C increased from 20 to 70%. Site-specific cleavage occurs, by up to 90%, at a single site of the target DNA. The melting temperature of the complementary complex formed by the bleomycin A5-modified tetranucleotide and target DNA in the presence of two effectors was 45 degrees C, whereas in the absence of the effectors, below 7 degrees C.

Antibiotics, Antineoplastic↗
PubMed1997

CpG methylation remodels chromatin structure in vitro.

One of the mechanisms proposed to explain how CpG methylation effects gene repression invokes a DNA methylation-determined chromatin structure. Previous work implied that this DNA modification does not influence nucleosome formation in vitro, thus current models propose that certain non-histone proteins or a preferential affinity by linker histones for methylated DNA may mediate changes in chromatin structure. We have reinvestigated whether CpG methylation alters the chromatin structure of reconstitutes comprising only core histones and DNA. We find that DNA methylation prevents the histone octamer from interacting with an otherwise high affinity positioning sequence in the promoter region of the chicken adult beta-globin gene. This exclusion is attributed to methylation-determined changes in DNA structure within a triplet of CpG dinucleotides. In the affected nucleosome, this sequence motif is located 1.5 helical turns from the dyad axis and is oriented towards the histone core. These findings establish that DNA methylation does have the capacity to modulate chromatin structure directly, at its most fundamental level. Furthermore, our observations strongly suggest that a very limited number of nucleotides can make a decisive contribution to the translational positioning of nucleosomes.

Animals↗
PubMed1986

In vivo metabolism and reaction with DNA of the cytostatic agent, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC).

The cytostatic drug dacarbazine [DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide] is strongly carcinogenic in rats. Bioactivation of DTIC yields a methylating intermediate but the extent of interaction with cellular macromolecules has not previously been reported. Following a single i.p. injection of [14C-methyl]DTIC, exhalation of 14CO2 occurred with a t1/2 max of approximately 2 hr (0.95 mg/kg) and 2.5 hr (95 mg/kg). Of the total radioactivity administered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predominantly as unchanged DTIC. In liver, kidney and lung, formation of 7-[14C]methylguanine in DNA and RNA was directly proportional with dose. DNA methylation by a single dose of DTIC (9.8 mg/kg; 5 hr survival time) was highest in liver (35 mumoles 7-methylguanine/mole guanine), followed by kidney (25 mumoles) and lung (20 mumoles). The remainder tissues showed 7-methylguanine concentrations approximately 50% of those in liver DNA, with the exception of the brain which had a very low extent of DNA modification (approximately 1 mumole/mole guanine). At the specific radioactivity used (48 mCi/mmole), the promutagenic base O6-methylguanine was only detectable in liver, kidney, lung, and stomach DNA (0.6-0.8 mumoles/mole guanine). Autoradiographic studies revealed a diffuse distribution of reaction products in rat liver. In contrast, N-nitrosodimethylamine and related carcinogens known to be bioactivated by the hepatic cytochrome P-450 system show a predominantly centrilobular distribution. This difference may be due to the greater stability of proximate carcinogens generated by alpha-C hydroxylation at one of the methyl groups of DTIC.

Animals↗
PubMed1987

Gene transfer from Escherichia coli to Campylobacter species: development of shuttle vectors for genetic analysis of Campylobacter jejuni.

A shuttle cloning vector (pIL550) has been constructed which can be mobilized from Escherichia coli to Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by complementation with the transfer functions of an IncP plasmid in trans, with a frequency of 10(-4) transconjugants per donor. We also present evidence for a DNA modification system in C. jejuni.

Campylobacter↗