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At least 811 records · Page 45Linked to original sources
PubMed1992

Effects of phenobarbital on I-compounds in liver DNA as a function of age in male rats fed two different diets.

The age-dependent effects of diet and of phenobarbital (PB), a known promoter of hepatocarcinogenesis, on indigenous DNA adducts (I-compounds) were studied by the 32P-post-labeling technique. Late-gestation female Sprague-Dawley rats were fed either AIN-76A semisynthetic or Teklad cereal-based diet. At 21 days after birth, the male pups were weaned and continued on the diets, with half of each dietary group receiving 0.05% PB mixed into the diet for 2, 4 and 8 months. Age-dependent increases in I-compound levels were observed. In addition, both the levels of individual I-compounds and the overall number of I-compounds were greater in rats fed the Teklad cereal-based diet than in those fed the AIN-76A diet. Independent of the parent diet, PB administration reduced the levels of the majority of I-compounds in a time-dependent manner. This effect of PB was detected earlier in the Teklad-fed than in the AIN-76A-fed group. In contrast to the I-compounds, a second group of spots, termed reverse I-compounds, declined between 2 and 4 months and, especially in AIN-76A-fed animals, tended to increase when PB was administered. It is hypothesized that alterations of DNA modification patterns may play a role in diet-modified hepatocarcinogenesis promoted by PB.

Age Factors↗
PubMed1995

UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes.

Protein-DNA interactions in mammalian cells can be analyzed at the nucleotide level of resolution by genomic sequencing techniques. The most sensitive genomic sequencing method uses the ligation-mediated polymerase chain reaction (LMPCR) for signal amplification to detect the positions of DNA modifications or strand breaks. Various probing methods are compatible with LMPCR, but dimethyl sulfate footprinting has most commonly been used. Here, we have examined the suitability of ultraviolet (UV) light as an in vivo footprinting agent to detect a wide variety of protein-DNA contacts. The distribution of the two major types of UV-induced DNA photoproducts (cyclobutane pyrimidine dimers and (6-4) photo-products) has been examined along the promoter sequences of three human genes. A comparison of UV-irradiated naked DNA and UV-irradiated cells reveals differences in the UV damage spectrum for both types of photoproducts. These differences can be either decreases or dramatic increases of photoproduct frequency. At the promoter of the c-jun gene, these differences ("photofootprints") co-localize with binding sites for two AP-1-like transcription factors, a CCAAT box binding protein, an SP-1 sequence, an NF-jun sequence, a related to serum response factor (RSRF) binding site and a sequence bound by an unknown factor. In the promoter of the gene coding for proliferating cell nuclear antigen (PCNA), photofootprints were seen at two SP-1 like sequences and two CCAAT boxes. The c-fos promoter is characterized by photofootprints at the serum response element (SRE), at the adjacent binding site for ternary complex factor (TCF), at an AP-1 site and at a binding site for a growth factor inducible protein (SIF). Photofootprints may be signatures of specific transcription factors or families of related factors since we noticed that the photofootprints seen at several common factor binding sites were similar or identical when the same site was analyzed in different genes. Photofootprints were not seen at sequences distant from transcription factor binding sites. A comparison of our UV photofootprinting data with data from experiments using other probing strategies shows that UV light has the potential to reveal all protein-DNA interactions provided there is a dipyrimidine sequence on either DNA strand within a factor binding site. The simplicity of using this probing agent together with its specificity for detecting a large variety of different factors should make UV light a generally useful tool for in vivo footprinting studies.

Base Sequence↗
PubMed2002

Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli.

8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.

Alkaline Phosphatase↗
PubMed1982

Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.

The function of lysyl residues of the sigma subunit of the RNA polymerase from Escherichia coli was investigated by chemical modification with trinitrobenzenesulfonic acid (TNBS). Following reaction with TNBS, analysis of the modified sigma indicated that trinitrophenylation was limited to the epsilon-amino groups of lysyl residues. Progressive loss in the activity of sigma followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of sigma activity. Kinetic analysis indicated that one lysyl group is critical for the function of sigma. TNP-sigma was able to form a holoenzyme complex with a binding affinity comparable to that of sigma. Promoter recognition studies were done by using HindIII fragments from T5 DNA. The TNP-sigma core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d(A-T)n and T7 DNA templates. TNP-sigma was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-sigma or sigma using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the conformation of sigma following trinitrophenylation.

DNA-Directed RNA Polymerases↗
PubMed1999

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.

Animals↗
PubMed2002

Acetylated, methylated, remodeled: chromatin states for gene regulation.

The activity state of a gene is determined not only by sequence-specific regulatory factors but also by a complex network of co-acting proteins. Many of these proteins directly or indirectly affect the structure of the chromatin environment into which the gene is embedded. Recent studies of histone and DNA modifications support and refine the concept of the chromatin environment being key to the establishment and maintenance of transcriptional activity or repression.

Acetylation↗
PubMed1991

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.

Bacillus thuringiensis↗
PubMed1978

Carcinogenicity of 4-nitrosoquinoline 1-oxide and its possible role in carcinogenesis by 4-nitroquinoline 1-oxide.

4-Nitrosoquinoline 1-oxide induced malignant tumors at the subcutaneous site of injection in mice. It affected Escherichia coli to induce the so-called UV-type lesion in cellular DNA. DNA base-quinoline adducts produced by the treatment of mammalian cellular DNA with this carcinogen were proved to be identical with those obtained by the action of 4-nitroquinoline 1-oxide. Although this carcinogen was reactive enough to modify DNA chemically by itself, a different DNA modification took place in a chemical process from thos obtained in the in vivo process.

4-Nitroquinoline-1-oxide↗
PubMed1999

Study on GMA-DNA adducts.

OBJECTIVE: DNA modification fixed as mutations in the cells may be an essential factor in the initiation step of chemical carcinogenesis. In order to explore the mechanism of gene mutation and cell transformation induced by glycidyl methacrylate (GMA), the current test studied the characteristics of GMA-DNA adducts formation in vitro. METHODS: In vitro test, dAMP, dCMP, dGMP, dTMP and calf thymus DNA were allowed to react with GMA (Glycidyl Methacrylate). After the reaction, the mixtures were detected by UV and subjected to reversed-phase HPLC on ultrasphere ODS reversed-phase column, the reaction products were eluted with a linear gradients of methanol (solvent A) and 10 mmol/L ammonium formate, pH 5.0 (solvent B). The synthesized adducts were then characterized by UV spectroscopy in acid (pH 1.0), neutral (pH 7.2), alkaline (pH 11.0) and by mass spectroscopy. RESULTS: The results showed that GMA could bind with dAMP, dCMP, dGMP and calf thymus DNA by covalent bond, and the binding sites were specific (N6 of adenine, N3 of cytosine). Meanwhile, a main GMA-DNA adduct in the reaction of GMA with calf thymus DNA was confirmed as N3-methacrylate-2-hydroxypropyl-dCMP. CONCLUSIONS: GMA can react with DNA and/or deoxynucleotide monophosphate and generate some adducts such as N6-methacrylate-2-hydroxypropyl-dAMP and N3-methacrylate-2-hydroxypropyl-dCMP, ets. Formation of GMA-DNA adducts is an important molecular event in gene mutation and cell transformation induced by GMA.

Animals↗
PubMed2002

Cancer epigenomics.

Research in cancer epigenomics is driven by the development of novel technologies and the utilization of model organisms ranging from yeasts to plants to vertebrates. For decades, the search for cancer genes has focused on genetic defects that were used as tags for identification of these genes. With the realization that epigenetic modifications, most importantly DNA methylation events, are frequently involved in transcriptional changes in both tumor suppressor genes and oncogenes, techniques have been developed that support the identification of novel cancer genes altered by DNA methylation alone or in combination with genetic events. Recent data demonstrate that, in addition to DNA methylation, chromatin modifications are also involved in gene regulation. We are now beginning to understand this interesting interplay between chromatin modifications, DNA methylation and gene regulation. This review will summarize our current knowledge of DNA methylation and histone modification in normal cells, introduce emerging concepts that show the intimate link between DNA methylation and chromatin modifications, and highlight recent advancements in our understanding of aberrant DNA methylation, with special emphasis on genome-wide hypermethylation.

DNA Methylation↗
PubMed2001

Topoisomerase I-mediated DNA damage.

Topoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions.

Animals↗
PubMed2002

Alkylation of guanine in DNA by S23906-1, a novel potent antitumor compound derived from the plant alkaloid acronycine.

The discovery of a new DNA-targeted antitumor agent is a challenging enterprise, and the elucidation of its mechanism of action is an essential first step in investigating the structural and biological consequences of DNA modification and to guide the rational design of analogues. Here, we have dissected the mode of action of the newly discovered antitumor agent S23906-1. Gel retardation experiments reveal that the diacetate compound S23906-1 and its monoacetate analogue S28687 form highly stable covalent adducts with DNA. The covalent adducts formed between S23906-1 and a 7-bp hairpin oligonucleotide duplex were identified by spectrometry. In contrast, the inactive compound S23907, lacking the two acetate groups of S23906-1, fails to yield covalent DNA adducts, indicating that the C1-C2 functionality is the DNA reactive moiety. DNase I footprinting and DNA alkylation experiments indicate that S23906-1 reacts primarily with guanine residues. A 30-mer oligonucleotide containing only G.C bp forms highly stable complexes with S23906-1 and S28687, whereas the equivalent A.T oligonucleotide is not a good substrate for these two drugs. The use of an oligonucleotide duplex containing inosines instead of guanosines identifies the guanine 2-amino group exposed in the minor groove of DNA as the potential reactive site. The reactivity of S23906-1 toward the guanine-N2 group was independently confirmed by fluorescence spectroscopy. Covalent DNA adducts were also identified in the genomic DNA of B16 melanoma cells exposed to S23906-1, and the specific accumulation of the drug in the nucleus of the cells was visualized by confocal microscopy. The elucidation of the mechanism of action of this highly potent anticancer agent opens a new field for future drug design.

Acronine↗
PubMed1996

Organ-specific oxidative DNA damage associated with normal birth in rats.

Mammalian DNA contains bulky endogenous DNA modifications (I-compounds), which increase with age in unexposed animals, as shown by 32P-postlabeling. We have examined the perinatal formation of a subclass (type II) of I-compounds in rat liver, kidney, skin and lung. These I-compounds represent bulky oxidative DNA lesions, defined herein as intrastrand base-base and base-sugar cross-links, adducts of lipid peroxidation products and DNA-protein cross-links. We observed a rapid increase in the levels of five bulky oxidative DNA lesions during the first hours after normal birth of rats, with total levels increasing 4.2-, 3.0- and 1.3-fold, respectively, in liver, kidney and skin. This effect was not noted in lung. The results were consistent with oxidative stress induced by the known sudden increase in partial oxygen pressure at birth in blood and tissues, implying inadequate antioxidant defenses in the affected neonatal organs. Hepatic oxidative damage appeared intensified by increased concentrations of pro-oxidants and reduced concentrations of antioxidants in the maternal diet. The postnatal DNA lesions are postulated to be premutagenic, as indicated by their bulky nature and persistence. Pathophysiological effects of oxidative DNA damage would be exacerbated by rapid cell proliferation in neonatal tissues and consequent fixation as mutations. In addition to inherited mutations, DNA lesions acquired as a consequence of normal birth may play a hitherto unrecognized role in spontaneous carcinogenesis and age-related degenerative diseases.

Animals↗
PubMed1997

Organ-specific oxidative DNA damage associated with normal birth in rats.

Mammalian DNA contains bulky endogenous DNA modifications (I-compounds), which increase with age in unexposed animals, as shown by 32P-postlabeling. We have examined the perinatal formation of a subclass (type II) of I-compounds in rat liver, kidney, skin and lung. These I-compounds represent bulky oxidative DNA lesions, defined herein as intrastrand base-base and base-sugar cross-links, adducts of lipid peroxidation products and DNA-protein cross-links. We observed a rapid increase in the levels of five bulky oxidative DNA lesions during the first hours after normal birth of rats, with total levels increasing 4.2-, 3.0- and 1.3-fold, respectively, in liver, kidney and skin. This effect was not noted in lung. The results were consistent with oxidative stress induced by the known sudden increase in partial oxygen pressure at birth in blood and tissues, implying inadequate antioxidant defenses in the affected neonatal organs. Hepatic oxidative damage appeared intensified by increased concentrations of pro-oxidants and reduced concentrations of antioxidants in the maternal diet. The postnatal DNA lesions are postulated to be premutagenic, as indicated by their bulky nature and persistence. Pathophysiological effects of oxidative DNA damage would be exacerbated by rapid cell proliferation in neonatal tissues and consequent fixation as mutations. In addition to inherited mutations, DNA lesions acquired as a consequence of normal birth may play a hitherto unrecognized role in spontaneous carcinogenesis and age-related degenerative diseases.

Animals↗
PubMed2004

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation.

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.

Alpha Particles↗
PubMed2006

Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance.

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.

Acetylation↗
PubMed2002

Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing.

During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic-apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.

Animals↗
PubMed2006

Photosensitization of biomolecules by phenothiazine derivatives.

It is well known that many drugs act as photosensitizers towards cells by interacting with various cellular components such as lipids, proteins and nucleic acids. The structural modifications of the cellular components may occur by direct interactions of the excited states (singlets or triplets) of the drugs with the biological substrate or indirectly, through reactive species of oxygen sensitised by the drug themselves. In particular, the phototoxic activity of various drugs correlated with their potential photomutagenic and photocarcinogenic effects, takes place through DNA modification. Phenothiazines, a class of antihistaminic (anti-H1) or neuroleptic drugs used in the therapy of mental illness, such as schizophrenia, organic psychoses and other mental disorders, are known to induce photosensitization of the skin by systemic use or by topical applications as antiallergic drugs. In this review we have focused our attention on the photosensitizing property of phenothiazines and related compounds both in vitro and in vivo systems. Particular attention has been given to the mechanism of photo reaction with biomolecules such as lipids, proteins and DNA. Moreover there is a growing interest in drugs having photobiological effects because of their possible application as phototherapeutics. It has been interesting in this context to mention briefly the possible application of phenothiazine derivatives as new photosensitizers for their therapeutic application in photodynamic therapy (PDT) or in the light inactivation of viruses and bacteria.

Animals↗