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At least 109 records · Page 6Linked to original sources
PubMed1990

Modification of DNA bases in mammalian chromatin by radiation-generated free radicals.

Modification of DNA bases in mammalian chromatin in aqueous suspension by ionizing radiation generated free radicals was investigated. Argon, air, N2O, and N2O/O2 were used for saturation of the aqueous system in order to provide different radical environments. Radiation doses ranging from 20 to 200 Gy (J.kg-1) were used. Thirteen products resulting from radical interactions with pyrimidines and purines in chromatin were identified and quantitated by using the technique of gas chromatography/mass spectrometry with selected-ion monitoring after acidic hydrolysis and trimethylsilylation of chromatin. The methodology used permitted analysis of the modified bases directly in chromatin without the necessity of isolation of DNA from chromatin first. The results indicate that the radical environment provided by the presence of different gases in the system had a substantial effect on the types of products and their quantities. Some products were produced only in the presence of oxygen, whereas other products were detected only in the absence of oxygen. Products produced under all four gaseous conditions were also observed. Generally, the presence of oxygen in the system increased the yields of the products with the exception of formamidopyrimidines. Superoxide radical formed in the presence of air, and to a lesser extent in the presence of N2O/O2, had no effect on product formation. The presence of oxygen dramatically increased the yields of 8-hydroxypurines, whereas the yields of formamidopyrimidines were not affected by oxygen, although these products result from respective oxidation and reduction of the same hydroxyl-adduct radicals of purines. The yields of the products were much lower than those observed previously with DNA.

Animals↗
PubMed1989

Metabolism and binding of benzo[a]pyrene in randomly-proliferating, confluent and S-phase human skin fibroblasts.

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-3H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.

Benzo(a)pyrene↗
PubMed1990

Repair of acetyl-aminofluorene modified pBR322 DNA in Xenopus laevis oocytes and eggs; effect of diadenosine tetraphosphate.

Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DNA repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coli. The repaired DNA is even efficiently replicated, the egg being "activated" by the microinjection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase alpha. However, the repair appears to be very limited, since it does not restore the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and/or to a very low level of DNA polymerase beta. This system was used to study the effect of diadenosine tetraphosphate (Ap4A) on DNA repair. Ap4A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap4A could be involved in switching off the replication machinery when DNA is badly damaged, thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.

2-Acetylaminofluorene↗
PubMed2025

100+ years of phase variation: the premier bacterial bet-hedging phenomenon.

Stochastic, reversible switches in the expression of Salmonella flagella variants were first described by Andrewes in 1922. Termed phase variation (PV), subsequent research found that this phenomenon was widespread among bacterial species and controlled expression of major determinants of bacterial-host interactions. Underlying mechanisms were not discovered until the 1970s/1980s but were found to encompass intrinsic aspects of DNA processes (i.e. DNA slippage and recombination) and DNA modifications (i.e. DNA methylation). Despite this long history, discoveries are ongoing with expansions of the phase-variable repertoire into new organisms and novel insights into the functions of known loci and switching mechanisms. Some of these discoveries are somewhat controversial as the term 'PV' is being applied without addressing key aspects of the phenomenon such as whether mutations or epigenetic changes are reversible and generated prior to selection. Another 'missing' aspect of PV research is the impact of these adaptive switches in real-world situations. This review provides a perspective on the historical timeline of the discovery of PV, the current state-of-the-art, controversial aspects of classifying phase-variable loci and possible 'missing' real-world effects of this phenomenon.

Gene Expression Regulation, Bacterial↗
PubMed1992

DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin.

The antineoplastic benzanthroquinone drug doxorubicin can undergo flavoenzyme-catalyzed one-electron reduction which, in an aerobic environment, leads to the generation of oxygen-derived species. We therefore sought to determine whether doxorubicin in the presence of NADH dehydrogenase and the transition metal ions Fe(III) or Cu(II) induces DNA base modifications in isolated human chromatin. NADH dehydrogenase-catalyzed reduction of doxorubicin (25-100 microM) caused hydroxyl radical production detected as methane generated from dimethyl sulfoxide; addition of isolated human chromatin to the system produced a concentration-dependent quenching of detectable hydroxyl radical formation. Doxorubicin (5-50 microM)-stimulated enzyme-catalyzed oxidation of NADH was also diminished, but still detectable, in the presence of chromatin. Doxorubicin-induced DNA base modifications in chromatin were measured by gas chromatography/mass spectrometry with selected-ion monitoring. Production of modified bases required the addition of transition metal ion and was enhanced by the addition of active flavoenzyme. The non-redox cycling analogue 5-iminodaunorubicin induced significantly less base modification than did doxorubicin. In the presence of Fe(III), NADH dehydrogenase-catalyzed reduction of doxorubicin caused enhancement in the content of all modified bases over control levels. Substitution of Cu(II) for Fe(III) altered both the degree and the pattern of doxorubicin/NADH dehydrogenase-induced base modifications. The scavengers of hydroxyl radical mannitol and dimethyl sulfoxide or catalase did not significantly affect doxorubicin/NADH/NADH dehydrogenase/transition metal ion-induced base modifications. Superoxide dismutase further enhanced production of all base modifications. The data demonstrate that flavoenzyme-catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, suggesting a possible mechanism for the mutagenicity of doxorubicin in vivo.

Chromatin↗
PubMed1992

DNA base modifications in chromatin of human cancerous tissues.

Free radical-induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady-state level of free radical-modified bases in cellular DNA. We have investigated endogenous levels of typical free radical-induced DNA base modifications in chromatin of various human cancerous tissues and their cancer-free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine-derived and six purine-derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected-ion monitoring. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack on DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts of modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified bases over the control levels. The results clearly indicate higher steady-state levels of modified DNA bases in cancerous tissues than in their cancer-free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.

Chromatin↗
PubMed1978

Bacteriophage Mu-induced modification of DNA is dependent upon a host function.

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.

Coliphages↗
PubMed1988

Regulation of mutator activities in maize.

We discuss the properties of the Mutator (Mu) transposable element family of maize. We report the cloning of bz2-mu1, a mutable allele containing a 1.4-kb Mu element, using a combination of transposon tagging and tests for differential hybridization to northern and Southern blots. We report the sequence of this allele and the Mu element insertion, and propose a model for the structure of the Bz2 locus. We discuss the relationship between increased DNA modification of Mu elements and loss of somatic instability at bz2-mu1. To further explore this aspect of regulation of Mutator, we have used gene-specific probes to determine the level of modification at this locus in active and inactive Mutator lines. We have also utilized CsCl density gradients to estimate the overall level of DNA modification in active and inactive lines; we find that Mu elements in active lines are hypomethylated relative to other maize nuclear DNAs examined, and that in inactive lines the level of modification in Mu elements is similar to the genome as a whole. Utilizing gamma-irradiation, we have demonstrated that inactive lines can be reactivated; this reactivation is first noted as restitution of the spotted kernel phenotype characteristic of bz2-mu1 in active Mutator lines. Hybridization analysis of DNA from reactivated plants demonstrates that the Mu elements in general, and specifically the Mu element at bz2-mu1, have the lower level of DNA modification characteristic of active lines. These results are discussed in terms of the role and timing of DNA modification in regulating Mutator activities.

Base Sequence↗
PubMed1988

Monitoring of interaction products of cis-diamminedichloroplatinum(II) and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) with DNA in cells from platinum-treated cancer patients.

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.

Carboplatin↗
PubMed1988

Modification of DNA ends and detection of restriction enzyme recognition sequences in their ligated junctions.

A program has been developed for the modelling of modifications in DNA ends, for the construction of ligated junctions, and for the analysis in these junctions of new restriction enzyme recognition sequences. This program allows the analysis of restriction enzyme specificities in ligated junctions of cohesive or blunt DNA ends. Cohesive ends are considered in their natural configuration or after modification by possible blunt-ending procedures. The program also allows the modelling of partial filling-in for 5'-single-stranded ends. This program has proven useful for the design of sequences with new restriction sites or to predict or confirm the sequence of junctions created by the ligation of modified ends.

Base Sequence↗
PubMed1975

Host specificity of DNA in haemophilus influenzae: The physiological and genetical bases of instability of restriction and modification of DNA in strain Rd.

Further investigations of the instability of restriction and modification properties of H. influenzae Rd strain were carried out. It has been shown that the instable properties of hsd Hind1 system are maintained even after transfer of this system to another H. influenzae strain. The expression of hsd Hind1 system is very sensitive to various physiological changes which do not influence the other hsd systems present in the same Rd strain. The instability of hsd Hind1 system is postulated to be connected with some regulator gene(s).

DNA Restriction Enzymes↗
PubMed1986

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene.

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.

Bacteriophage phi X 174↗
PubMed1990

Age-dependent covalent DNA alterations (I-compounds) in rat liver mitochondrial DNA.

Rat liver mitochondrial (mt) DNA was investigated for the presence of I-compounds, a recently discovered type of DNA modifications which is detected and measured via 32P-postlabeling. These DNA modifications were previously shown to accumulate in an age-dependent manner in total cellular DNA of various tissues of untreated rodents. In the present work, mt DNA of 1-, 3-, 6-, and 9-month-old female Sprague-Dawley rats was found by 32P-postlabeling also to contain I-compounds that increase with age. Most of the I-compounds were identical for mt and nuclear (nu) DNA. A cluster of 2 non-polar I-spots (termed M-compounds) was mitochondria-specific and increased about 8-fold from 1 to 9 months, attaining a RAL value of 44 X 10(-9) or 1 modification in 2.3 X 10(7) DNA nucleotides at 9 months. Quantitative differences between chromatographically identical spots were seen mainly for a low-polarity fraction of I-compounds, which exhibited 2 times higher overall levels in mt DNA versus nu DNA over the age range studied. Total I-compound levels increased during this time 6.9- and 5.1-fold in nuclei and mitochondria, respectively. The M-compound level was close to 10% of total mt DNA I-compound levels. M-compounds may conceivably be derived from potentially DNA-reactive electron carriers of the mt electron-transport chain, while I-compounds common to both mt and nu DNA presumably originate in extramitochondrial sources. The similarity of mitochondrial and nuclear I-compound profiles and amounts implies possible regulatory mechanisms in I-compound formation and repair. Mt DNA maps showed additional 32P-labeled material which may have been associated with DNA damage caused by oxygen free radicals known to be generated by the mt electron-transport chain. Age-dependent increases of mt DNA modifications are potentially related to mt mutations and may be linked to age-related degenerative changes in mitochondria.

Aging↗
PubMedDate not supplied

[Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides].

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.

Autoradiography↗
PubMed1990

Biophysical studies of the modification of DNA by antitumour platinum coordination complexes.

Cisplatin (cis-diamminedichloroplatinum(II] is widely used in the treatment of various human tumours. A large body of experimental evidence indicates that the reaction of cisplatin with DNA is responsible for the cytostatic action of this drug. Several platinum-DNA adducts have been identified and their effect on the conformation of DNA has been investigated. Structural studies of platinum-DNA adducts now permit a reasonably good explanation of the biophysical properties of platinated DNA. Antitumouractive platinum compounds induce in DNA, at low levels of binding, local conformational alterations which have the character of non-denaturing distortions. It is likely that these changes occur in DNA due to the formation of intrastrand cross-links between two adjacent purine residues. On the other hand, the modification of DNA by antitumour-inactive complexes results in the formation of more severe local denaturation changes. Conformational alterations induced in DNA by antitumour-active platinum compounds may be reparable with greater difficulty than those induced by the inactive complexes. Alternatively, non-denaturation change induced in DNA by antitumour platinum drugs could represent more significant steric hindrance against DNA replication as compared with inactive complexes.

Animals↗
PubMed1978

Modification of DNA by aflatoxin B1 creates alkali-labile lesions in DNA at positions of guanine and adenine.

The damage to DNA by the hepatocarcinogen aflatoxin B1 was investigated. A DNA fragment of known sequence of the lactose promoter-operator region was used as a substrate for modification by aflatoxin B1. The DNA was incubated with aflatoxin B1 in crude mammalian liver extracts or with purified microsomes. Treatment of the DNA incubated in the complete system with either 1 M piperidine or 0.1 M NaOH at 90 degrees revealed alkali-labile lesions in the DNA. The exact location of the cleavage site was determined by comparison of the length of the cleavage products with the known sequence on polyacrylamide gels. The lengths of the cleavage products were the same as those produced by alkali-induced breakage of the same sequence of DNA that had been modified with dimethyl sulfate. The major cleavage products of the aflatoxin B1-modified DNA were at positions of guanine and the minor cleavage products were at positions of adenine. These studies show that modification of DNA by aflatoxin B1 creates alkali-labile sites at positions of guanine and, to a lesser extent, adenine.

Aflatoxins↗
PubMed1978

Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: relation to chromatin structure and reconstitution.

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S1 than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the 'open' regions of chromatin, and 35% to 'closed' regions. These results indicate that although convalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.

Animals↗
PubMedDate not supplied

DNA methylation and epigenetic mechanisms.

Genes are essential for the transmission of genetic information from generation to generation, and this mechanism of inheritance is fully understood. Genes are also essential for unfolding the genetic program for development, but the rules governing this process are obscure. Epigenetics comprises the study of the switching on and off of genes during development, the segregation of gene activities following somatic cell division, and the stable inheritance of a given spectrum of gene activities in specific cells. Some of these processes may be explained by DNA modification, particularly changes in the pattern of DNA methylation and the heritability of that pattern. There is strong evidence that DNA methylation plays an important role in the control of gene activity in cultured mammalian cells, and the properties of a CHO mutant strain affected in DNA methylation are described. Human diploid cells progressively lose cytosine methylation during serial subculture, and this may be related to their in vitro senescence. There is also evidence that DNA modifications can be inherited through the germ line. Classical genetics is based on the study of all types of change in DNA base sequence, but the rules governing the activity of genes by epigenetic mechanisms are necessarily different. Their elucidation will depend both on a theoretical framework for development and on experimental studies at the molecular, chromosomal, and cellular levels.

Animals↗