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At least 613 records · Page 34Linked to original sources
PubMed1991

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.

Animals↗
PubMed2004

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes.

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a water-soluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkali-labile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

Animals↗
PubMed2004

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine.

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1983

Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation.

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.

DNA↗
PubMed2005

Human cytochrome P450 reductase can act as a source of endogenous oxidative DNA damage and genetic instability.

Studies with repair-deficient mice and other experiments suggest that oxidative DNA modifications are generated in all types of cells even under physiological conditions and that this type of endogenous DNA damage contributes to spontaneous cancer incidence. However, the cellular sources of reactive oxygen species that are relevant for nuclear oxidative DNA damage are largely unknown. Here, we report that expression of human NADPH-cytochrome P450 reductase (hOR) in cultured V79 Chinese hamster cells gives rise to elevated basal levels of oxidative purine modifications after depletion of glutathione. Also, the basal levels of micronuclei are increased in the hOR-expressing cells, and again the effect is enhanced when the antioxidant defense system of the cells is diminished by depletion of glutathione. The oxidative DNA damage is increased when duroquinone, a substrate of hOR, is added, both in the presence and absence of glutathione. In contrast, hOR-expressing cells are similarly sensitive as the parental cells when oxidative DNA damage and micronuclei are induced by a mechanism independent of hOR, i.e., exposure to bromate. The results identify hOR as a potential source of endogenous oxidative DNA damage and subsequent genetic instability in mammalian cells.

Animals↗
PubMed2007

REBASE--enzymes and genes for DNA restriction and modification.

REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in the biological process of restriction-modification. It contains fully referenced information about recognition and cleavage sites, isoschizomers, neoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. Experimentally characterized homing endonucleases are also included. All newly sequenced genomes are analyzed for the presence of putative restriction systems and these data are included within the REBASE. The contents or REBASE may be browsed from the web (http://rebase.neb.com/rebase/rebase.ftp.html) and selected compilations can be downloaded by ftp (ftp.neb.com). Additionally, monthly updates can be requested via email.

DNA Modification Methylases↗
PubMed2005

The repair of DNA damages/modifications during the maturation of the immune system: lessons from human primary immunodeficiency disorders and animal models.

The immune system is the site of various genotoxic stresses that occur during its maturation as well as during immune responses. These DNA lesions/modifications are primarily the consequences of specific physiological processes such as the V(D)J recombination, the immunoglobulin class switch recombination (CSR), and the generation of somatic hypermutations (SHMs) within Ig variable domains. The DNA lesions can be introduced either by specific factors (RAG1 and RAG2 in the case of V(D)J recombination and AID in the case of CSR and SHM) or during the various phases of cellular proliferation and cellular activation. All these DNA lesions are taken care of by the diverse DNA repair machineries of the cell. Several animal models as well as human conditions have established the critical importance of these DNA lesions/modifications and their repair in the physiology of the immune system. Indeed their defects have consequences ranging from immune deficiency to development of immune malignancy. The survey of human pathology has been highly instrumental in the past in identifying key factors involved in the generation of DNA modifications (AID for the Ig CSR and generation of SHM) or the repair of specific DNA damages (Artemis for V(D)J recombination). Defects in factors involved in the cell cycle checkpoints following DNA damage also have deleterious consequences on the immune system. The continuous survey of human diseases characterized by primary immunodeficiency associated with increased sensitivity to ionizing radiation should help identify other important DNA repair factors essential for the development and maintenance of the immune system.

Animals↗
PubMed2005

DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters.

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.

Archaeal Proteins↗
PubMed2006

Novel bifunctional viologen-linked pyrene conjugates: Synthesis and study of their interactions with nucleosides and DNA.

With the objective of developing efficient DNA oxidizing agents, a new series of viologen-linked pyrene conjugates with the general formula PYLnV(2+), having a different number of methylene spacer units (Ln) was synthesized, and their interactions with nucleosides and DNA have been investigated through photophysical and biophysical techniques. The viologen-linked pyrene derivatives PYL1V(2+) (n =equals; 1), PYL7V(2+) (n = 7), and PYL12V(2+) (n = 12) exhibited characteristic fluorescence emission of the pyrene chromophore centered around 380 nm but with significantly reduced yields when compared to those of the model compound PYL1Et(3)(+). The fluorescence quenching observed in these systems is explained through an electron-transfer mechanism based on a calculated favorable change in free energy (DeltaG(ET) = -1.59 eV), and the redox species characterized through laser flash photolysis studies. Intramolecular electron-transfer rate constants (k(ET)) were calculated from the observed fluorescence yields, and the singlet lifetimes of the model compound and are found to decrease with increasing spacer length. The DNA binding studies of these systems through photophysical, chiroptical, and viscometric techniques demonstrated that these systems effectively undergo DNA intercalation with association constants (KDNA) in the range of 1.1-2.6 x 10(4) M(-1) and exhibit 2:1 sequence selectivity for poly(dG) x poly(dC) over poly(dA) x poly(dT). Photoactivation of these systems initiates electron transfer from the singlet excited state of the pyrene chromophore to the viologen moiety followed by an electron transfer from DNA to the oxidized pyrene. This results in the formation of stable charge-separated species such as radical cations of both DNA and reduced viologen as characterized by laser flash photolysis studies and subsequently the oxidized DNA modifications. These novel systems are soluble in buffer media, stable under irradiation conditions, and oxidize DNA efficiently and selectively through a cosensitization mechanism and hence can be useful as photoactivated DNA cleaving agents.

Animals↗
PubMed1998

Partial characterization of two major liver I-compounds as unstable adducts which are readily hydrolyzed to unmodified guanine nucleotides.

I-compounds are endogenous bulky DNA modifications which are detected by nuclease P1-enhanced 32P-post-labeling in tissue DNA of animals not knowingly exposed to carcinogens. Their profiles and levels depend inter alia on animal age, species, strain, tissue, gender, diet and exposure to chemicals such as cytochrome P450 inducers and carcinogens. Due to lack of sufficient material obtainable from in vivo sources, chemical structures of I-compounds and their parent normal bases have not yet been identified. In this report we provide 32P-post-labeling and chromatographic evidence that two prominent I-compounds, herein called C1 and C2, which occur at relatively high levels in pig liver DNA are guanine derivatives. This result was obtained by showing that both compounds, isolated from 32P-post-labeling thin-layer maps, were chemically unstable, i.e. they could be readily hydrolyzed to 32P-post-labeled deoxyguanosine 3',5'-bisphosphate by heating in water. C1 appeared particularly labile, undergoing hydrolysis during thin-layer chromatography at pH 3.3 without heating. Several other I-compounds and adducts, as well as the four normal DNA nucleotides, were, however, highly resistant to hydrolysis under the conditions used here. The possible significance of these findings will be briefly discussed.

Animals↗
PubMed2005

Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.

Amino Acid Sequence↗
PubMed1982

DNA methylation in eukaryotes.

The DNA of higher eukaryotes contains one minor base, namely 5-methylcytosine. The distribution of this minor base between different species and different DNA fractions will be considered together with the actual sequences methylated. The properties of the enzyme responsible for DNA modification will be reviewed, particular note being paid to the efficiency of methylation of different DNA substrates. Various possible functions of the 5-methylcytosine in DNA will be considered and particular attention will be paid to the finding that specific modified bases present in DNA not undergoing transcription are absent in the same genes when these are being actively transcribed.

5-Methylcytosine↗
PubMed2002

[Utility of oxidative stress test in the male infertility clinic].

The controlled generation of very low amounts of reactive oxygen species (ROS) appears to regulate normal sperm functions, while high levels of ROS endanger sperm viability and function. Oxidative stress (OS) develops as a consequence of excessive production of ROS and/or impaired antioxidant defense system. It is proposed that such OS precipitates a range of pathologies currently thought to afflict male reproductive function. ROS-mediated peroxidative damage to the sperm plasma membrane may account for defective sperm function observed in a high proportion of infertility patients. Excessive generation of ROS may also attack integrity of DNA in the sperm nucleus. DNA bases are susceptible to oxidative stress, and peroxidation of these structures can cause base modification, DNA strand breaks and chromatin cross-linking. DNA damage induced by excessive ROS may accelerate the process of germ cell apoptosis, leading to decline in sperm counts associated with male infertility, and may explain the apparent deterioration of semen quality observed during the past four to five decades. For almost a decade, our research team in the Cleveland Clinic Foundation has identified the critical role of OS in male infertility. The main objective of our research was to transfer this important knowledge from the research bench to clinical practice. We designed studies with the aims of: 1. understanding the exact mechanisms by which OS develops in semen, which we thought will help setup strategies to overcome the problem, 2. establishing assays for accurate assessment of OS status and running the quality control studies for this purpose, 3. testing the correlation between OS and sperm nuclear DNA damage, and 4. identifying the clinical significance of seminal OS assessment in male infertility practice.

DNA↗
PubMed1988

Potential anti-tumour 2,5-bis (1-aziridinyl)-1,4-benzoquinones: relationship between cytotoxicity against DNA-repair deficient E. coli, inactivation of bacteriophage M13mp19, and in vitro and in vivo anti-tumour activity.

A series of reductively activated 3,6 substituted 2,5-bis(aziridinyl)benzoquinones, designed as potential anti-tumour agents, was shown to interact with DNA. This was assayed by studying the colony forming ability of DNA-repair deficient E. coli K12 and the inactivation of bacteriophage M13mp19. The activities of the compounds as measured in the two assays show a good correlation. Reduction of the quinone compounds is essential for activity in the two assays. Reduction activates opening of the aziridine ring and formation of an alkylating species. The results suggest that steric factors are important in DNA adduct formation. Methylation of the aziridine rings decreases the activity. Bulky 3,6-substitutes also decrease the activity. No correlation is found between above-mentioned assays for DNA modification and in vivo activity towards tumour cells. It is possible that, as well as DNA, other cellular targets are involved. A correlation is observed between the DNA modifying activity and toxicity against L1210 cells in vitro. The relative high activity against L1210 cells of bromine substituted compounds is possibly related to alternative mechanisms in which bromine acts as an efficient leaving group.

Animals↗
PubMed1988

[Cloning of the restriction-modification genes of Bacillus centrosporus in Escherichia coli].

A genomic library of Bacillus centrosporus was obtained using pBR327 as a vector. The total plasmid DNA of the library was cleaved by the BcnI restriction endonuclease and then transformed in Escherichia coli RR1. Two clones possessing restriction and DNA modification profiles of BcnI were identified among the transformants. Their respective plasmids were 13.3 and 9.05 kbp in size. Restriction mapping of both plasmids showed each of them to contain two sites for HindIII and one for both Eco31I and Eco47III, located at the same distance. This was assumed to be the location region of the BcnI restriction-modification genes. Confirmation of the assumption was obtained by deletion mapping of the recombinant plasmids. Special features concerning cloning of the restriction-modification genes are discussed on the basis of the results obtained.

Bacillus↗
PubMedDate not supplied

[Complementary addressed modification of plasmid DNA].

The possibility to accomplish the sequence-specific chemical modification of superhelical DNA with reactive oligonucleotide derivatives was demonstrated. Plasmids containing fragments of the immunoglobulin gene were modified with alkylating derivatives of oligonucleotides complementary to a nucleotide sequence in the immunoglobulin gene. In contrast to the relaxed plasmid DNAs, superhelical DNAs (sigma = -0.1) were found to be attacked by the derivatives at the target nucleotide sequence. The efficiency of the reaction increases with the increase of the plasmids negative superhelicity. It was found also that the denatured derivatives. The sequence-specific modification of plasmid DNAs with the reactive oligonucleotide derivatives can be used for the site-directed mutagenesis and the investigation of the repair processes.

Animals↗
PubMed1975

The effect of modification of T7 DNA by the carcinogen N-1-acetylaminofluorene: termination of transcription in vitro.

To study the effects of N-2-acetylaminofluorene (AAF) modification of DNA on transcription, purified DNA from bacteriophage T7 was modified in vitro to varying extent with AAF and transcribed by DNA-dependent RNA polymerase from Escherichia coli. The main effects of AAF modification on transcription are a marked inhibition of the rate and extent of trna synthesis with relatively little effect on initiation except at very high AAF doses. Calibration of the percent modification with [14-C]AAF and analysis of the size of the RNA product by double isotope labeling and polyacrylamide gel electrophoresis support the following mechanism of transcription inhibition: most of the AAF residues bound to the coding strand of the DNA cause premature termination of transcription, at or near the site of modification, with release of RNA polymerase. This results in the production of shorter RNA chains with increasing amounts of bound carcinogen. The data are consistent with there being no reinitiation and/or synthesis of RNA distal to the AAF-modification site.

Carcinogens↗
PubMed1980

Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light.

Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac)2ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%-1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations. DNA species exhibiting, in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%-1.9% agarose gels, the decrease os superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%-0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges.

2-Acetylaminofluorene↗