Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Modifications”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed2001

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference.

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

Adenine↗
PubMed1995

E-box variants direct formation of distinct complexes with the basic helix-loop-helix protein ALF1.

The murine transcription factor ALF1 belongs to the class of basic helix-loop-helix proteins specific for the NCAGNTGN-version of the E-box. Binding of homodimeric ALF1 to variants of this motif was studied by a combination of binding site selection technology and DNA modification interference analysis. The results showed that substitutions at the non-conserved positions in the E-box sequence could cause profound alterations in the patterns of specific contacts at the protein-DNA interface. Thus, both the overall extent of the binding region and the backbone phosphate contact pattern differed markedly between closely related E-boxes with similar affinities for ALF1. The identity of the base at the inner N was an important determinant of contact pattern specification. The E-box variants differed in their ability to mediate ALF1 dependent transcriptional activation in vivo. We discuss the possibility that adaptability in basic helix-loop-helix protein-DNA interactions can result in complexes with different functional properties.

Amino Acid Sequence↗
PubMed1976

A protective effect of caffeine on the ethidium induced petite mutation in yeast.

A prerequisite for petite induction by ethidium bromide (EB) is an initial covalent attachment of the drug to cytoplasmic DNA. This DNA modification is thought to initiate repair processes. The repair inhibitor, caffeine, provided a protective effect against the ethidium induced petite mutation at caffeine concentrations known to inhibit the repair of UV damage in cytoplasmic DNA (Fig. 1). Mitochondrial DNA isolated from yeast exposed to EB in vivo was not as degraded in the presence of both drugs as with EB alone (Fig. 2).

Caffeine↗
PubMed1979

Is the DNA of virus T7 methylated?

DNA modification of T7 wild type and of T7 M-mutants was studied by determining the percentage of 5-methylcytosine (5MC)/cytosine (C) and N6-aminopurine (6MA)/adenine (A) and by evaluating the plating efficiencies of restriction-sensitive T7 M-mutants on modifying and non-modifying host strains. Only 0.03% adenine and 0.02% cytosine were methylated in the DNA of T7 wild type as well as in T7 M-mutants, which was independent of the host DNA methylation (30- to 50-fold higher). The restriction of T7 M-mutants, determined from the plating efficiencies, was not altered on modifying or non-modifying hosts. These results indicate that host-specific modification is blocked during T7 development and that this is not due to the M-protein.

Adenine↗
PubMed1997

Analysis of cytogenetic effects and DNA adduct formation induced by safrole in Chinese hamster lung cells.

Safrole (1-allyl-3,4-methylenedioxybenzene) was tested for its ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) and to form DNA adducts in Chinese hamster lung (CHL) cells, in order to investigate the relationship between cytogenetic effects and DNA adduct formation under the same treatment conditions. The cells were treated with 0.025-0.2 mg/ml safrole in the presence or absence of rat liver postmitochondrial supernatant fraction (S9). Safrole induced significant SCEs and CAs dose-dependently in the presence of S9. SCEs ranged in number from 15.6 to 21.1 SCEs/cell and CAs were observed in 4-37% of cells. Using the 32P-postlabeling assay, two major and two minor safrole-DNA adducts were detected in DNA digests obtained from CHL cells in the presence of S9. The levels of total DNA adducts ranged from 1.3 to 22.8 adducts/10(7) nucleotides. The two major adducts were shown to be guanine derivatives since these adducts comigrated on polyethylenimine plates with the adducts produced by the reaction of safrole with 2'-deoxyguanosine 3'-monophosphate. A correlation was seen between DNA adducts and SCEs or CAs. Neither induction of SCEs and CAs nor formation of DNA adducts was observed in the absence of S9. These findings suggest that SCEs and CAs induced by safrole result from covalent DNA modification metabolically activated by S9 in cultured cells.

Animals↗
PubMed1987

DNA methylation in rat tissues by a series of homologous aliphatic nitrosamines ranging from N-nitrosodimethylamine to N-nitrosomethyldodecylamine.

Aliphatic N-nitrosomethylalkylamines exhibit a remarkable organ specificity in rats, the principal targets for tumour induction being liver, oesophagus, urinary bladder and lung. We have determined the extent of DNA methylation in these tissues following a single oral dose (0.1 mmol/kg; 6 h survival) of each of 12 homologues, ranging from N-nitrosodimethylamine (C1) to N-nitrosomethyldodecylamine (C12). Methylpurines (7- and O6-methylguanine) were determined by cation exchange HPLC with fluorescence detection. Highest levels of hepatic DNA methylation were found with N-nitrosodimethylamine (C1) and N-nitrosomethylethylamine (C2), the most potent hepatocarcinogens in this series. Concentrations of methylpurines in liver DNA decreased with increasing chain length for C1-C5. Administration of the higher homologues (C6-C12) caused levels of DNA methylation which by themselves were considered too low to account for their hepatocarcinogenicity. In rat oesophagus, DNA methylation closely paralleled carcinogenicity, the butyl and pentyl derivatives (C4, C5) being most effective. In rat lung, the extent of DNA methylation was generally lower and there was no apparent correlation with carcinogenicity. Methylation of kidney DNA also decreased with increasing chain length and was only detectable for C1-C5. In urinary bladder DNA, methylpurines were below or close to the limit of detection. It is concluded that the initiation of malignant transformation by DNA methylation alone (through hydroxylation at the methylene alpha-carbon) could be operative for C1 in kidney and lung, for C1 and C2 in liver, and C3-C5 in oesophagus. For the higher homologues, the extent of DNA methylation seems insufficient to explain the complex pattern of tissue specificity, suggesting that DNA modification other than, or in addition to, methylation may be responsible.

Animals↗
PubMed1994

Unusual properties of genomic DNA molecules spanning the euchromatic-heterochromatic junction of a Drosophila minichromosome.

While investigating the copy number of minichromosome Dp(1;f)1187 sequences in the polyploid chromosomes of ovarian nurse and follicle cells of Drosophila melanogaster we discovered that restriction fragments spanning the euchromatic-heterochromatic junction of the chromosome and extending into peri-centromeric sequences had the unusual property of being selectively resistant to transfer out of agarose gels during Southern blotting, leading to systematic reductions in Dp1187-specific hybridization signals. This property originated from the peri-centromeric sequences contained on the junction fragments and was persistently associated with Dp1187 DNA, despite attempts to ameliorate the effect by altering experimental protocols. Transfer inhibition was unlikely to be caused by an inherent physical property of repetitive DNA sequences since, in contrast to genomic DNA, cloned restriction fragments spanning the euchromatic-heterochromatic junction and containing repetitive sequences transferred normally. Finally, the degree of inhibition could be suppressed by the addition of a Y chromosome to the genotype. On the basis of these observations and the fact that peri-centromeric regions of most eukaryotic chromosomes are associated with cytologically and genetically defined heterochromatin, we propose that peri-centromeric sequences of Dp1187 that are incorporated into heterochromatin in vivo retain some component of heterochromatic structure during DNA isolation, perhaps a tightly bound protein or DNA modification, which subsequently causes the unorthodox properties observed in vitro.

Animals↗
PubMedDate not supplied

Age-dependent increase of indigenous DNA adducts in rat brain is associated with a lipid peroxidation product.

Indigenous DNA adducts (I-compounds) are considered to be a biomarker of aging tissues. Thus far, few studies have been conducted to investigate the accumulation patterns of I-compounds in the brain during aging. Particularly, identities of age-dependent I-compounds have largely remained unknown. In the current study, we have determined the amounts of I-compounds in the brains of male Fischer 344 rats at ages 1, 6, 12, 18, and 24 months using a 32P-postlabeling technique. The results indicate that I-compounds increase in the rat brain age dependently from 6 to 24 months of age. Total I-adduct levels (central and upper cutouts) increase 3.5-fold from 6 to 24 months. Contrary to the results of other investigators, brains of 1-month-old rats contain the highest level of I-compounds, which may be due to the hypermetabolic status during the infant period. In an effort to characterize I-compounds, different deoxynucleosides were coincubated with malondialdehyde (MDA). The results show that only deoxyguanosine (dGMP)-MDA adducts overlap with I-compounds of the rat brain DNA adducts map. A total of five dGMP-MDA adducts have been identified as responsible for I-compounds in brain tissues. It is known that brain tissue contains high levels of lipids that are susceptible to oxygen free radicals and that MDA is the most abundant and genotoxic product of lipid peroxidation. The present study provides supporting evidence that lipid peroxidation and its product (MDA) may play an important role in endogenous brain DNA modification, which may partly contribute to cerebral aging and age-related degenerative disorders of the brain. The accumulation of I-compounds with aging may serve as an index of indirect oxidative damage to DNA as evidenced by the presence of MDA-DNA adducts.

Aging↗
PubMed2003

Benzo[a]pyrene enhances lipid peroxidation induced DNA damage in aorta of apolipoprotein E knockout mice.

The genotoxic compound benzo[a]pyrene (B[a]P) enhances atherosclerotic plaque progression, possibly by inducing oxidative stress and subsequent lipid peroxidation (LPO). Since LPO plays a key role in atherosclerosis, stable LPO derived DNA modifications such as 1,N6-ethenodeoxy-adenosine (epsilondA) and 3,N4-ethenodeoxy-cytidine (epsilondC) may be useful biomarkers for in vivo oxidative stress. In this study, benzo[a]pyrene-diol-epoxide (BPDE)-DNA, epsilondA and epsilondC were determined by 32P-postlabelling in apolipoprotein E knockout (ApoE-KO) mice treated with 5mg/kg B[a]P by gavage. After 4 days, BPDE-DNA adduct levels were higher in aorta (10.8 +/- 1.4 adducts/10(8) nucleotides) than in lung (3.3 +/- 0.7, P < 0.05), which is a known target organ for B[a]P. Levels of epsilondA were higher in aorta of B[a]P-exposed animals than in unexposed controls (8.1 +/- 4.4 vs 3.4 +/- 2.1 adducts per 10(8) parent nucleotides, P < 0.05). On the other hand, epsilondC levels were not affected by B[a]P exposure. Serum low density lipoprotein (LDL) levels were lower in B[a]P-exposed mice than in controls (9.3 +/- 3.7 and 13.3 +/- 4.0mmol/l, respectively), whereas high density lipoprotein (HDL) levels were higher (1.4 +/- 1.6 and 0.4 +/- 0.3mmol/l, respectively). Consequently, a three-fold difference in the LDL/HDL ratio was observed (P = 0.001). epsilondA levels were positively related with plasma HDL concentrations (R = 0.68, P = 0.02), suggesting that the HDL mediated protection of the vessel wall against reactive lipid peroxides was reduced in B[a]P-exposed apoE-KO mice. Our observations show that direct as well as lipid peroxidation induced DNA damage is formed by B[a]P in aorta of apoE-KO mice, which may be involved in atherosclerotic plaque progression. This study further indicates that etheno-DNA adducts are useful biomarkers for in vivo oxidative stress in atherosclerosis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Metabolic activation of 6-nitrochrysene in explants of human bronchus and in isolated rat hepatocytes.

It has previously been shown that 6-nitrochrysene can be activated to electrophilic species capable of reacting with DNA through metabolic pathways that form N-hydroxy-6-aminochrysene or trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as critical intermediates. Since the lung is a known target tissue for the carcinogenic action of polycyclic nitroaromatic hydrocarbons, we investigated the metabolism and DNA binding of [3H]6-nitrochrysene in 11 specimens of human bronchus. Analysis of medium from [3H]6-nitrochrysene-treated explants indicated the presence of trans-9,10-dihydroxy-9,10-dihydro-6-nitrochrysene (0.04-330 pmol/mg epithelial DNA), trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (12-1700 pmol/mg epithelial DNA), 6-aminochrysene (1.6-2200 pmol/mg epithelial DNA), and trans-1,2-dihydroxy-1,2-dihydro-6-aminochyrsene (3.6-610 pmol/mg epithelial DNA). Both the levels and the relative proportions of these metabolites varied widely in explants from different individuals. The amount of DNA recovered and the level of DNA modification were sufficient for adduct analysis in eight of the 11 cases for which metabolite data were obtained. Five additional bronchial specimens for which metabolite data were not obtained were also analyzed for carcinogen-DNA adducts. The levels of binding varied from 0.06 to 30.5 pmol [3H]6-nitrochrysene bound/mg DNA (two adducts per 10(8) nucleotides-10 adducts per 10(6) nucleotides). HPLC analyses of enzymatic hydrolysates of the explant DNA indicated that 11 of 13 cases contained adducts with retention times identical to those of adducts derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene or N-hydroxy-6-aminochrysene. The adduct derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene was the major adduct detected in eight of 13 cases. The reasons for the variation in metabolism and adduct formation observed in [3H]6-nitrochrysene-treated explants of bronchus from different donors are not known but may reflect differences in the activities of enzymes responsible for the metabolism of this compound. The influence of induction of drug metabolizing enzymes on the activation pathway of 6-nitrochrysene in an intact cell system was tested using rat hepatocytes. 6-Nitrochrysene was incubated with freshly isolated hepatocytes from rats that were either untreated or pretreated with phenobarbital, 3-methylcholanthrene or Aroclor 1254. Although the levels of adducts were similar in all cases, the pattern of DNA adducts formed in these hepatocytes was dependent on the nature of the pretreatment of the rats. As previously reported, hepatocytes from untreated rats contained adducts derived from N-hydroxy-6-aminochrysene.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Immunological detection and quantification of carcinogen-modified DNA components.

Both the detection and quantitation of defined reaction products of chemical carcinogens with DNA, especially at low levels of DNA modification and in small numbers of target cells, require highly sensitive analytical techniques. The sensitivity of radiochromatographic methods is limited by the specific radioactivity of the respective carcinogens and by the relatively large amounts of DNA required for analysis. Furthermore, their application is restricted to experiments with radiolabelled carcinogens synthesized in the laboratory. These shortcomings can be circumvented by the use of high-affinity antibodies specifically directed against DNA components structurally modified by carcinogens, in combination with sensitive immunoassay procedures. Such immunological methodology, using both conventional antisera and monoclonal antibodies, has recently become available and is rapidly being developed further. We review here the properties of the antibodies thus far produced, the techniques applied for their development and characterization, and the immunoassay procedures currently in use. Some examples of the application of the new immunoanalytical methods are given.

Antibody Specificity↗
PubMed1998

[Extrachromosomal genetic elements of Clostridium botulinum. II. Isolation and analysis of DNA from bacteriophages of Clostridium botulinum types C and A].

The DNAs of bacteriophage c-st, known to realize the lysogenic conversion of toxinogenicity among C. botulinum types C and D strains, and the nucleic acid of a virulent mutant of bacteriophage CB propagated in type A C. botulinum cells were purified and examined. Heterogeneity of phage c-st preparations was observed during purification, manifesting by formation of several bands in isopiknic CsCl gradient during centrifuging. An extra nucleic acid fraction was detected in some DNA preparations of phage c-st; the origin of this fraction is discussed. Plasmid extrachromosomal elements were for the first time found in the cells of nontoxigenic type C C. botulinum A02 strain, known as the indicator for c-st phage. The sensitivity of phage c-st DNA to 25 restriction endonucleases was examined. Analysis of the results of restriction analysis of c-st and CB phage DNAs and plasmid nucleic acids, revealed earlier in type A C. botulinum strains, disclosed several DNA modification enzymes with different recognition sites in type C C. botulinum. At least two of these activities are not found in type A strains. According to restriction analysis, total size of phage c-st DNA is about 160 kbp and of phage CB DNA 35 kbp. Individual EcoRI and HindIII restricts of phage c-st DNA, containing the initial site of botulinum toxin CI gene, were recognized by radioisotope labeled oligonucleotide probe Enzyme immunoassay revealed slight expression of the N-terminal region of bntc I gene in E. coli recombinant variants. These data can be used in further investigation of C. botulinum genetics.

Bacteriophages↗
PubMed2005

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases.

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B-Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B-Z junction as revealed by X-ray crystallography at 2.6 A resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

Base Pairing↗
PubMed2002

Laser mediated production of reactive oxygen and nitrogen species; implications for therapy.

Laser therapy has gained wide acceptance applications to many medical disciplines. The side effect-effects from laser therapy involve the potential for interaction with cellular and extracellular matrix molecules to generate reactive oxygen species and reactive nitrogen species which in turn can initiate lipid peroxidation, protein damage or DNA modification. These issues are addressed in this short overview in the context of experimental models of laser-induced thrombosis.

Animals↗
PubMed1993

Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters.

Reactive oxygen species (ROS) have been implicated as being involved in tumor promotion processes. However, the mechanism by which ROS modulate tumor promotion has not as yet been elucidated. In this report, we show that phorbol ester-type tumor promoters (12-O-tetradecanoylphorbol-13-acetate [TPA], mezerein and 12-O-retinoylphorbol-13-acetate [RPA]), which vary in their in vivo potencies, also differ in their effect on formation of hydrogen peroxide (H2O2) and oxidation of normal bases to 5-hydroxymethyl-2'-deoxyuridine [HMdU] and 8-hydroxyl-2'-deoxyguanosine [8-OHdG] in the DNA of SENCAR mouse epidermis, though they are equipotent in causing infiltration of polymorphonuclear leukocytes (PMNs). Treatment of SENCAR mice with the chemopreventive agents (-)-epigallocatechin gallate or tamoxifen (6.5 nmol) prior to application of TPA (6.5 nmol) diminished PMN infiltration, and formation of H2O2, HMdU and 8-OHdG. These results strengthen the evidence that ROS are involved in tumor promotion, and that generation of ROS and the subsequent oxidative DNA modification are related to the tumor-promoting potencies of the different phorbol ester-type promoters.

Animals↗
PubMed1999

Propagation of a novel cytoplasmic, infectious and deleterious determinant is controlled by translational accuracy in Podospora anserina.

Some mutant strains of the filamentous fungus Podospora anserina spontaneously present a growth impairment, which has been called Crippled Growth (CG). CG is caused by a cytoplasmic and infectious factor, C. C is efficiently transmitted during mitosis but is not transmitted to the progeny after sexual crosses. C is induced by stationary phase and cured by various means, most of which stress the cells. Translational accuracy is shown to tightly regulate the propagation of C during the active growth period, because its propagation in dividing hyphae is restricted to cells that display an increased translational accuracy. However, induction of C in stationary phase proceeds independently from the translational accuracy status of the strain. CG does not seem to be accompanied by mitochondrial DNA modifications, although C activates the action of the Determinant of Senescence, another cytoplasmic and infectious element, which causes a disorganization of the mitochondrial genome. In addition, presence of C drastically modifies the spectrum of the mitochondrial DNA rearrangements in AS6-5 mat- cultures during Senescence. C seems to belong to the growing list of unconventional genetic elements. The biological significance of such elements is discussed.

Ascomycota↗
PubMed1999

[DNA repeats and homologous recombination: a probable role for DNA methylation in genome stability of eukaryotic cells].

Homologous recombination between DNA repeats directly threatens the intact transmission of repeat-rich eukaryotic genomes through mitotic and meiotic cell divisions. Besides several other factors already known, DNA methylation might contribute, in some eukaryotes, to the limitation of crossover events between repeats. A strong inhibitory effect of DNA methylation has now been directly demonstrated, in the filamentous fungus Ascobolus. This therefore reinforces the question of the biological impact of this DNA modification on the recombinational stability of repeat-rich genomes, such as those of mammals.

Animals↗
PubMed1982

Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine.

Treatment of Friend erythroleukemia cells with the antileukemic drugs 5-azacytidine and 5-aza-2'-deoxycytidine leads to rapid, time-dependent, and dose-dependent decrease of DNA methyltransferase activity and synthesis of markedly undermethylated DNA. Since this DNA is at least partially methylated in vivo and serves as an excellent substrate for methylation in vitro, hypomethylation of DNA in analog-treated cells appears to result from the loss of DNA methyltransferase, rather than from an inherent inability of 5-azacytosine- substituted DNA to serve as a methyl acceptor. Inhibition of DNA synthesis blocks the loss of DNA methyltransferase activity while inhibitors of RNA synthesis do not, suggesting that the analogs must be incorporated into DNA to mediate their effect on the enzyme, and that minor substitution of 5-azacytosine for cytosine in DNA (approximately 0.3%) suffices to inactivate more than 95% of the enzyme in the cell. Several lines of evidence link changes in the pattern of DNA modification with differentiation. In this regard, it is significant that 5-azacytidine and 5-aza-2'-deoxycytidine act as weak inducers of erythroid differentiation of Friend erythroleukemia cells in the same concentration range where they affect DNA methyltransferase activity. For differentiation to proceed, the cells must be washed free of the drugs. Less than 24 h later, normal levels of DNA methyltransferase activity are restored and within 48 h, DNA isolated from the cells is not detectably undermethylated. This may in part explain why 5-azacytidine and 5-aza-2'-deoxycytidine induce differentiation in less than 15% of the population despite their initial profound effect on DNA methylation.

Animals↗