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At least 829 records · Page 46Linked to original sources
PubMed2002

Efficiency of Iε promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightly linked to the strength of transcription.

One key unresolved issue in immunoglobulin class switch recombination (CSR) is how the accessibility of the switch region for CSR is controlled. To better understand the nature of accessibility control for human Ig CSR, we developed a novel inducible switch recombination assay based on expression of green fluorescence protein (GFP) from switch constructs undergoing substrate switch recombination (SSR). Efficient SSR depends on the cytokine-inducible Iepsilon promoter and co-stimulation with IL-4+anti-CD40. Characterization of SSR reveals that both S-S deletional recombination and S-S inversion occur. We show that the IL-4-inducible Iepsilon promoter (pIepsilon) selectively determines the efficiency of the accessibility for SSR. However, the pIepsilon-induced transcription, by itself,is not sufficient to direct efficient SSR. For efficient SSR, both pIepsilon-driven transcriptional activity and an additional promoter/enhancer-derived activity are required. The efficiency of SSR is not tightly correlated with the strength of the combined transcriptional activity. Our results suggest that the mechanism(s) underlying the transcriptional activity, e.g. DNA modification is important for controlling the accessibility for efficient switch recombination.

Animals↗
PubMed1995

A novel repetitive sequence lies near the gene encoding a cytosine methyltransferase in the cyanobacterium Dactylococcopsis salina.

An unusual cluster of tandemly repeated DNA sequences (TRS) was found downstream from the gene encoding DsaV methyltransferase, the DNA modification enzyme in the DsaV restriction-modification system found in a strain of Dactylococcopsis salina (Ds). The repeat unit is about 32-bp long and is present 13 times in the cluster. Each repeat unit can be divided into two distinct parts based on the level of sequence conservation and evolution. Hybridization of Ds DNA with a probe specific for this cluster revealed that there were at least two additional sites within the genome with similar TRS. The TRS units are localized in one region of the Ds genome. They do not share significant sequence similarity with other TRS found in prokaryotes.

Base Composition↗
PubMed2003

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein.

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.

Adenosine Triphosphate↗
PubMed1990

Low-dose-rate effect of tritium beta-rays on transfection activity of phage DNA related to an oxidative species different from .OH, HO2. O2-., and H2O2.

In order to study the low-dose-rate effect of tritium beta-irradiation, we exposed single-stranded DNA of M13 mp10 phage to purified tritiated water. The beta-irradiated DNA was then transfected to E. coli K12 JM101. The lower the radiation dose rate, the more the transfection activity decreased. After the low-dose-rate effect was confirmed, the characteristics of tritiated water were also studied. The formation of an active oxidative species different from .OH, HO2., O2-., and H2O2 was found in the tritiated water by using UV spectrometry and luminescence measurement. Attributing it to nescent oxygen by reason of the production of the epoxide from mesityl oxide, we discussed the low-dose-rate effect from the viewpoint that the yield of the DNA modification could be relatively higher than that of the O2 formation in a competition reaction when the dose rate was lower.

Bacteriophages↗
PubMed1996

DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Animals↗
PubMed2001

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyguanosine.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) was allowed to react in the presence of 2'-deoxyguanosine (16 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC/MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 24% of 1 reacted with different heteroatoms of the nucleoside. As expected, the principal site of 2'-deoxyguanosine alkylation was N7. Alkylation of N7 caused spontaneous depurination, and N-(7-guaninylethyl)-N-hydroxyethyl-p-aminophenylbutyric acid (5) and the corresponding N7,N7-bis-adduct (6) were the major stable dGuo derivatives. Also several other adducts were detected and tentatively identified by means of MS/MS and UV. From them, the O(6-), N1-, N(2-), and O5'-derivatives can be biologically significant. Our results shed new light on DNA modifications caused by chlorambucil, which is an important chemotherapeutic drug and a known carcinogen.

Antineoplastic Agents, Alkylating↗
PubMed1999

Site-specific DNA methylation and apoptosis: induction by diabetogenic streptozotocin.

Streptozotocin (STZ) is known to induce insulin-dependent diabetes mellitus via DNA damage in experimental animals. The mechanism of induction of DNA damage by STZ was investigated in vitro, using a human cell line and 32P-labeled DNA fragments isolated from human genes. STZ induced cellular DNA damage and apoptosis, and frequently initiated DNA modification at guanines, especially at the middle guanine in runs of three and at the guanine at the 3'-end of runs of two guanines, similar to N-methyl-N-nitrosourea, a typical methylating agent. Scavengers for reactive oxygen species or nitric oxide did not inhibit the induction of DNA damage by STZ. On the other hand, damage induction was inhibited by sodium acetate and sodium chloride, which can reduce the reactivity of methylating agents to DNA via the sodium cation. These results suggest that STZ induces DNA damage by methylation of guanines via methyl cations. This alkylation may be responsible for triggering apoptosis, and subsequently diabetes.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

The scope of dansyl vs fluorescein label in fluorescence postlabeling assay for DNA damage.

The 5'-phosphate groups of the normal nucleotides of DNA (10 nmol) were labeled with two different fluorescent reagents, namely, dansyl chloride and fluorescein-isothiocyanate, via phosphoramidate derivatives of ethylene and hexamethylenediamines. Using a conventional detector, HPLC analysis showed a linear relationship of fluorescence signal as an integrated peak area and the amounts of nucleotide injected from 2 pmol to 200 fmol (r = 0.996, n = 3) for dansyl-labeled dAmp and from 2 pmol to 90 fmol (r = 0.997) for fluorescein-labeled dAmp, respectively. Although fluorescein-labeled nucleotides are more sensitive than dansylated nucleotides (detection limit < 2 times, S/N = 3), the excess labeling reagent from the fluorescein labeling reaction interferes with HPLC resolution of the labeled nucleotides. The phosphoramidate derivatives of the nucleotides require purification prior to labeling with fluorescein isothiocyanate. As a result, the overall assay procedure becomes complicated. The potential of dansylated nucleotides to assay DNA modifications has been demonstrated by HPLC resolution of the labeled nucleotides of both normal and modified bases without further manipulation of the reaction intermediates.

Chromatography, High Pressure Liquid↗
PubMed1989

Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells.

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.

Alkylating Agents↗
PubMed2004

Cell survival, DNA, and protein damage in B14 cells under low-intensity near-infrared (810 nm) laser irradiation.

OBJECTIVE: The aim of this study was to reveal the possible cytotoxic and genotoxic effects of low-intensity (200 mW) near-infrared (810 nm) laser irradiation, using B14 cell line. BACKGROUND DATA: Laser therapy is widely used in biomedical treatment of many diseases, but the possible molecular mechanisms of laser actions remain unclear and the damaging effects of laser irradiation are still controversial. The side effects of laser therapy involve the generation of reactive oxygen and nitrogen species which in turn initiate lipid peroxidation, protein damage or DNA modification. METHODS: B14 cells and suspension of human erythrocyte membranes were irradiated with near-infrared (810 nm) therapy laser at different radiant exposures (3.75-15.0 J/cm(2)) and light power (fluency rate) 200 mW at 22 degrees C. Laser induced cellular oxidative damage was measured in terms of cell survival, DNA damage, measured using the method of single cell gel electrophoresis (Comet assay), protein damage measured as protein carbonyls formation. RESULTS: No substantial changes of cell survival under B14 cells irradiation at radiant exposures 3.75-11.25 J/cm(2) were observed. Similarly, neither considerable light-induced DNA damage or protein carbonyls accumulation was revealed. On the contrary, laser irradiation has led to decrease of cell protein carbonyl groups level in a dose-dependent manner. Additionally, using human red blood cell membranes as model membranes and biological oxidant HOCl we observed that laser irradiation resulted in a decrease of the level of membrane protein carbonyl groups accumulated under oxidative HOCl treatment. CONCLUSIONS: We can conclude that laser irradiation used (810 nm, 200 mW, 3.75-11.25 J/cm(2)) did not produce any considerable cytotoxic or genotoxic effects in B14 cells. Moreover, laser irradiation reduced cellular protein damage (protein carbonyl groups) produced by biological oxidant HOCl.

Animals↗
PubMed1997

Effects of uracil incorporation, DNA mismatches, and abasic sites on cleavage and religation activities of mammalian topoisomerase I.

Abasic sites and deamination of cytosine to uracil are probably the most common types of endogenous DNA damage. The effects of such lesions on DNA topoisomerase I (top1) activity were examined in oligonucleotides containing a unique top1 cleavage site. The presence of uracils and abasic sites within the first 4 bases immediately 5' to the cleavage site suppressed normal top1 cleavage and induced new top1 cleavage sites. Uracils immediately 3' to the cleavage site increased cleavage and produced a camptothecin mimicking effect. A mismatch with a bulge or abasic sites immediately 3' to the top1 cleavage site irreversibly trapped top1 cleavable complexes in the absence of camptothecin and produced a suicide cleavage complex. These results demonstrate that top1 activity is sensitive to physiological, environmental, and pharmacological DNA modifications and that top1 can act as a specific mismatch- and abasic site-nicking enzyme.

Animals↗
PubMed2003

Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand.

Somatic hypermutation and class switch recombination are DNA modification reactions that alter the genes encoding antibodies in B lymphocytes. Both of these distinct reactions require activation-induced deaminase (AID) and transcription. Here we show that in Escherichia coli, as in eukaryotic cells, the mutation frequency is directly proportional to the transcription of target genes. Transcription enhances mutation of the nontemplate DNA strand, which is exposed as single-stranded DNA during the elongation reaction, but not mutation of the template DNA strand, which is protected by E. coli RNA polymerase. Our results establish a direct link between AID and transcription and suggest that the role of transcription in facilitating mutation is to provide AID with access to single-stranded DNA.

Animals↗
PubMed2005

The amazing complexity of transcription factories.

Multicellular eukaryotes orchestrate immensely complex patterns of gene expression in order to define numerous specialised cell types. Patterns of expression are regulated at the level of DNA modification, chromatin structure, genome architecture and nuclear organisation. Each aspect of these features of regulation is controlled by complex nuclear systems, and each of the systems must interact to define a coherent regulatory network. In proliferating cells, and particularly during development, patterns of gene expression must be preserved in cells that have active DNA repair mechanisms and efficiently duplicate both DNA and the associated chromatin status--the histone code. The systems that regulate DNA synthesis and genome stability are integrated with those that regulate the efficacy of RNA synthesis, such that patterns of gene expression are maintained. This review analyses how different experimental approaches are revealing the amazing complexity of the transcriptionally active compartment of mammalian cells. The functional implications of this complexity will be discussed.

Animals↗
PubMed2000

DNA-adduct levels as a predictor of outcome for NSCLC patients receiving daily cisplatin and radiotherapy.

We aimed to investigate whether biological factors related to radiosensitivity and chemosensitivity have prognostic significance in non-small-cell-lung-cancer (NSCLC) patients treated with daily low doses of cisplatin and radiotherapy. We treated 27 NSCLC patients with concomitant daily low-dose cisplatin and radiotherapy between 1993 and 1995. Tumour specimens were analyzed for p53 and bcl-2 expression, and for cell proliferation using antibodies against ki-67. In addition, apoptosis was measured by an end-labeling technique (TUNEL). Finally, cisplatin-induced DNA modification in buccal cells was assessed immunocytochemically using a specific anti-serum. Univariate and multivariate analyses were performed to assess the association between the different variables and survival. The median follow-up was 41 months, and 21 patients (78%) have died. In a univariate analysis, age, tumour stage and cisplatin-DNA-adduct staining were the only factors significantly associated with survival (p < 0.05, log-rank test). p53, bcl-2, Ki-67 and apoptosis showed no relationship with outcome. Multivariate analysis revealed that cisplatin-DNA-adduct staining remained an independent prognostic factor (hazard ratio, 0.10, 95% CI, 0.02-0.49), with shorter survival times for patients with low adduct staining.

Adult↗
PubMed2005

Replication of heterochromatin: insights into mechanisms of epigenetic inheritance.

Heterochromatin is composed of tightly condensed chromatin in which the histones are deacetylated and methylated, and specific nonhistone proteins are bound. Additionally, in vertebrates and plants, the DNA within heterochromatin is methylated. As the heterochromatic state is stably inherited, replication of heterochromatin requires not only duplication of the DNA but also a reinstallment of the appropriate protein and DNA modifications. Thus replication of heterochromatin provides a framework for understanding mechanisms of epigenetic inheritance. In recent studies, roles have been identified for replication factors in reinstating heterochromatin, particularly functions for origin recognition complex, proliferating cell nuclear antigen, and chromatin-assembly factor 1 in recruiting the heterochromatin binding protein HP1, a histone methyltransferase, a DNA methyltransferase, and a chromatin remodeling complex. Potential mechanistic links between these factors are discussed. In some cells, replication of the heterochromatin is blocked, and in Drosophila this inhibition is mediated by a chromatin binding protein SuUR.

Animals↗
PubMed1990

Strain differences of I-compounds in relation to organ sites of spontaneous tumorigenesis and non-neoplastic renal disease in mice.

Possible associations between the patterns and levels of I-compounds (putative endogenous, age-dependent, adduct-like DNA derivatives) and the incidence of spontaneous tumors and renal diseases in mice were investigated by 32P-postlabeling assay. Liver, lung and kidney DNAs of 8-10 month old inbred male mice of strains C57BL, C3H, CBA, A and RFM were examined. It was found that liver DNA of the C3H mouse, known to develop a high incidence of spontaneous hepatomas, and lung DNA of the A mouse, known to have a high incidence of spontaneous lung tumors, contained smaller numbers and significantly lower total levels of I-compounds as compared with the resistant C57BL mouse. Kidney DNA of C57BL, C3H and A mice presented significantly higher levels of I-compounds compared with those of CBA and RFM mice, both of which are known for exceptionally high incidences of spontaneous renal diseases. Total kidney I-compound levels in CBA and RFM mice were only 30-40% of those found in C3H, A and C57BL. Reduced I-compound levels observed in the three organs thus tended to be associated with spontaneous tumorigenesis and degenerative disease in susceptible strains, suggesting that I-compounds may be important for normal gene transcription and DNA replication. On the other hand, some of these DNA derivatives may, as adduct-like premutagenic lesions, contribute to spontaneous tumorigenesis. This was suggested by the finding that CBA mice which exhibit moderate spontaneous hepatoma incidence, had higher levels of certain I-compounds in their liver DNA although the total levels were not significantly different compared with those in C57BL mice. The observations of the present study suggest that (i) the patterns and levels of I-compounds are genetically determined and (ii) lack or excess of these DNA modifications may result in adverse health effects; however, this may not always be the case, for example, if modulating factors exist which compensate for I-compound deficiency.

Age Factors↗
PubMed1998

In vivo genotoxicity and DNA adduct levels in the liver of rats treated with safrole.

The induction of chromosome aberrations, sister chromatid exchanges (SCEs), and the formation of DNA adducts was studied in hepatocytes of F344 rats exposed in vivo to safrole. Hepatocytes were isolated 24 h after a single dose of safrole or five repeated doses (once a day) by gastric intubation and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after 48 h in culture. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-post-labeling assay in isolated hepatocytes from the rats. While a single dose was not sufficient to induce detectable levels of chromosome aberrations at the time of assay, five repeated doses induced these changes with a maximum frequency of 13.4%, compared with the control value of 1.8%. Both a single dose and five repeated doses induced significant SCEs, to a maximum frequency of 0.81 SCEs per chromosome, while the control value was 0.59 SCEs per chromosome. Two major and two minor DNA adducts were detected after treatment with either a single dose or five repeated doses. The maximum amount of total DNA adducts was 89.8 DNA adducts/10(7) nucleotides. These results show that safrole is a genotoxic carcinogen in the rat liver in vivo and suggest that the cytogenetic effects of this compound may result from covalent DNA modification in the rat liver. This in vivo cytogenetic assay should provide a useful means of evaluation of the genotoxicity of hepatocarcinogens.

Animals↗
PubMed1987

Development of a monoclonal antibody-based immunoassay for cyclic DNA adducts resulting from exposure to crotonaldehyde.

In order to develop an immunoassay for DNA modifications resulting from exposure to crotonaldehyde, monoclonal antibodies specific for the 8R,6R- and 8S,6S-stereoisomers of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxy-6 - methylpyrimido[1,2-a]purine-10(3H)one were produced. These cyclic 1,N2-propanodeoxyguanosines are formed in DNA exposed to crotonaldehyde in vitro. Three of the four antibodies were most specific for one stereoisomer while the fourth was most specific for the other stereoisomer. Fifty % inhibition of binding in an enzyme-linked immunoabsorbent assay using two of these antibodies and capable of detecting 0.5 mumol of 1,N2-propanodeoxyguanosine per mol of deoxyguanosine was developed. The method was validated by comparison to results obtained with fluorescence assay.

Aldehydes↗