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At least 1,225 records · Page 68Linked to original sources
PubMed1994

Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae.

The gene encoding the XorII methyltransferase (M.XorII) was cloned from Xanthomonas oryzae pv. oryzae and characterized in Escherichia coli. The M.XorII activity was localized to a 3.1 kb BamHI-BstXI fragment, which contained two open reading frames (ORFs) of 1272 nucleotides (424 amino acids) and 408 nucleotides (136 amino acids). Ten polypeptide domains conserved in other M5 cytosine methyltransferases (MTases) were identified in the deduced amino acid sequence of the 1272 ORF. E. coli Mrr+ strains were transformed poorly by plasmids containing the XorII MTase gene, indicating the presence of at least one MCG in the recognition sequence for M.XorII (CGATCG). The 408 nucleotide ORF was 36% identical at the amino acid level to sequences of the E. coli dem-vsr gene, which is required for very short patch repair. X. oryzae pv. oryzae genomic DNA that is resistant to digestion by PvuI and XorII hybridizes with a 7.0 kb fragment containing the XorII MTase gene and vsr homolog, whereas DNA from strains that lack M.XorII activity do not hybridize with the fragment.

Amino Acid Sequence↗
PubMed2006

Crystal structure of SUMO-3-modified thymine-DNA glycosylase.

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular beta-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM.

Amino Acid Sequence↗
PubMed2001

Emerging connections between DNA methylation and histone acetylation.

Modifications of both DNA and chromatin can affect gene expression and lead to gene silencing. Evidence of links between DNA methylation and histone hypoacetylation is accumulating. Several proteins that specifically bind to methylated DNA are associated with complexes that include histone deacetylases (HDACs). In addition, DNA methyltransferases of mammals appear to interact with HDACs. Experiments with animal cells have shown that HDACs are responsible for part of the repressive effect of DNA methylation. Evidence was found in Neurospora that protein acetylation can in some cases affect DNA methylation. The available data suggest that the roles of DNA methylation and histone hypoacetylation, and their relationship with each other, can vary, even within an organism. Some open questions in this emerging field that should be answered in the near future are discussed.

Acetylation↗
PubMed1999

[New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase].

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.

Azides↗
PubMed1988

Sequence-specific chemical modification of double-stranded DNA with alkylating oligodeoxyribonucleotide derivatives.

Chemical modification of double-stranded (ds) DNA with alkylating oligodeoxynucleotide (oligo) derivatives, 5'-p(N-2-chloroethyl-N-methylamino) benzylamides of oligos, has been investigated. In contrast to relaxed plasmid DNAs, the superhelical molecules interact with the oligo derivatives and specific alkylation of the DNAs occurs at the regions complementary to the oligo reagents. Alkylating derivatives of oligocytidylates and pT(pCpT)6 react with corresponding homopyrimidine-homopurine tracts within ds DNA fragments due to triple helix formation.

Alkylation↗
PubMed1992

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.

Animals↗
PubMed1983

Induction of the base displacement or Z conformation in DNA by N-2-acetylaminofluorene modification.

Modification of deoxyguanosine at the C(8) position by the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) has been shown to result in two different conformational changes dependent on the nucleotide sequence of the modified polymer. AAF modification of random sequence DNA results in a large distortion of the helix which is termed base displacement. In this conformation, the carcinogen is inserted into the DNA perpendicular to the helix axis with the guanosine displaced to the outside. Large single-stranded regions are generated which are susceptible to S(1) nuclease digestion and react with anti-cytidine antibodies.A different conformation has been observed when the alternating purine pyrimidine copolymer, poly(dG-dC).poly(dG-dC) is modified. At a modification level of 28% this polymer shows a CD spectrum characteristic of the left-handed Z-DNA seen in the unmodified polymer at high ethanol or salt concentrations. Base pairing of the modified polymer remains intact as demonstrated by its resistance to digestion with S(1) nuclease and lack of reactivity with anti-cytidine antibodies. Modification of poly(dG-m(5)dC).poly(dG-m(5)dC) with AAF was also shown to induce the Z conformation. However, for this polymer, inversion of the CD spectrum takes place at a much lower modification level (10%) than for the nonmethylated polymer (>20%). This polymer is also resistant to S(1) nuclease digestion consistent with its adoption of the Z conformation with AAF modification. A possible role in gene expression for the Z conformation of AAF modified regions is discussed.

2-Acetylaminofluorene↗
PubMed1989

[Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6].

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.

Alkylating Agents↗
PubMed1990

Role of glutathione in the radiation response of mammalian cells in vitro and in vivo.

Radiation interacts with biological systems to produce many types of molecular lesions. Much of the molecular damage is of little consequence with regard to cell killing. The lesions that are most likely to contribute to cell killing are DNA lesions produced by clusters of radicals. The formation of clusters of radicals is characteristic of ionizing radiation and accounts for its high efficiency as a cytotoxic agent. The mechanism by which these lesions kill cells is probably the formation of DNA double-strand breaks, ultimately resulting in chromosomal breaks. There is a possibility that some of the other types of molecular lesions produced by radiation may participate in more subtle mechanisms of cell damage. For instance, radiation induces a self-destructive process (apoptosis) in certain cell types, and the molecular lesions that initiate this process have not been identified. Glutathione (GSH) is a versatile protector. Several distinct mechanisms of radioprotection by GSH can be identified. These include radical scavenging, restoration of damaged molecules by hydrogen donation, reduction of peroxides and maintenance of protein thiols in the reduced state. Of these mechanisms, hydrogen donation to DNA radicals is probably the most important. Since competing reactions are very rapid, this mechanism requires a high concentration of GSH. Radioprotection by hydrogen donation to DNA radicals is not effective in oxygenated cells because the normal intracellular GSH concentration is not sufficient for effective competition with oxygen. Consequently, moderate depletion of GSH has no effect on the radiosensitivity of oxygenated cells. Under hypoxic conditions GSH becomes more competitive, and GSH depletion can markedly affect radiosensitivity. The radiosensitivity of hypoxic cells is most affected by GSH depletion in the presence of low concentrations of radiosensitizers. Since hypoxic cells are a characteristic feature of tumors, moderate depletion of GSH in combination with treatment with hypoxic cell radiosensitizers appears to be a promising strategy for selective tumor sensitization in radiation therapy. Oxidation of GSH can result in radiosensitization of both hypoxic and oxygenated cells. The mechanism of this effect appears to involve oxidation of protein thiols which are important for DNA repair. In principle, modification of DNA repair could have a greater impact on radiation therapy than modification of the number of lesions produced by radiation. However, a strategy for modification of GSH or protein thiol redox state in vivo has not yet been devised.

Animals↗
PubMed2005

Controlling DNA methylation: many roads to one modification.

Genetic, biochemical and cytological studies on DNA methylation in several eukaryotic organisms have resulted in leaps of understanding in the past three years. Discoveries of mechanistic links between DNA methylation and histone methylation, and between these processes and RNA interference (RNAi) machineries have reinvigorated the field. The details of the connections between DNA methylation, histone modifications and RNA silencing remain to be elucidated, but it is already clear that no single pathway accounts for all DNA methylation found in eukaryotes. Rather, different taxa use one or more of several general mechanisms to control methylation. Despite recent progress, classic questions remain, including: What are the signals for DNA methylation? Are "de novo" and "maintenance" methylation truly separate processes? How is DNA methylation regulated?

Chromatin↗
PubMed1979

An enzyme-linked immunosorbent assay for antibodies to native and denatured DNA.

A modification of the enzyme-linked immunosorbent assay (ELISA) is described, that permits determination of antibodies to native DNA (nDNA). The same approach can be used to measure antibodies to denatured DNA (dDNA). Poor binding of nDNA to the polystyrene solid phase has presented difficulties in using the ELISA method for assaying anti-nDNA activity (Engvall, 1976), but we find that precoating of the solid phase with protamine sulfate circumvents this problem. Assays for anti-dDNA are also enhanced by the use of protamine sulfate coated tubes. We have used the ELISA method to assay 15 SLE and 27 non-SLE sera for anti-nDNA and anti-dDNA activity. The results are compared with those obtained using the GF/A glass fiber filter assay, previously described by Lewis et al. (1973).

Animals↗
PubMed1998

DNA interactions of antitumor trans-[PtCl2(NH3)(quinoline)].

Recent observations that several trans-platinum complexes exhibit antitumor activity including activity in cisplatin-resistant tumor cells, violates the classical structure/activity relationships of platinum(II) complexes. According to these relationships, only bifunctional platinum(II) complexes with cis-oriented leaving ligands should be therapeutically active. In order to contribute to the understanding of mechanisms underlying the antitumor activity of these new trans-platinum analogs, various biochemical and biophysical methods as well as molecular modeling techniques were employed to study the modifications of DNA by antitumor trans-[PtCl2(NH3)(quinoline)]. The results indicated that trans-[PtCl2(NH3)(quinoline)] coordinated monofunctionally to DNA with a similar rate as transplatin. The overall rate of the rearrangement to bifunctional adducts was also similar to that observed in the case of DNA modification by transplatin, i.e. it was relatively slow (after 48 h approximately 34% adducts remained monofunctional). In contrast to transplatin, however, trans-[PtCl2(NH3)(quinoline)] formed considerably more interstrand cross-links after 48 h (approximately 30%) with a much shorter half-time (approximately 5 h) (approximately 12% for transplatin, t1/2 > 11 h). The results also suggested that the quinoline ligand in all or in a significant fraction of DNA adducts of trans-[PtCl2(NH3)(quinoline)], in which platinum is coordinated to base residues, was well positioned to interact with the duplex. The adducts of trans-[PtCl2(NH3)(quinoline)] terminated in vitro RNA synthesis preferentially at guanine residues. Surprisingly, the type and extent of conformational alterations induced in DNA indicates that trans-[PtCl2(NH3)(quinoline)] behaves in some respects like cisplatin, as indicated by the fact that trans-[PtCl2(NH3)(quinoline)]-modified DNA is recognized by cisplatin-specific antibodies. Models for both monofunctional adducts and bifunctional interstrand cross-links are proposed. Computer-generated AMBER models show that the combination of monofunctional covalent binding and a stacking interaction between quinoline and the DNA bases can produce a kink in the duplex which is strongly suggestive of the directed bend produced by the major cisplatin-DNA adduct (1,2 intrastrand cross-link). Unique DNA adducts of this type formed by trans-[PtCl2(NH3)(quinoline)] may contribute to the antitumor efficacy of this agent.

Animals↗
PubMedDate not supplied

[DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins].

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

Cysteine↗
PubMedDate not supplied

[Application of alkylating DNA derivatives for addressed modification of the genome].

To affect definite preselected regions of DNA, complementary denaturated DNA fragments carrying alkylating groups were used. The residues of polyfunctional alkylating agent N',N'N'-tri(beta-chloroethyl)-N'-(p-formylphenyl)propylenediamine-1,3 were attached covalently to 4-5% of bases of the T7 phage DNA (T7 DNA) restriction fragment. The alkylating DNA derivatives was found to be stable under hybridisation conditions. It was shown that the DNA fragment carrying alkylating groups is capable of highly specific interaction with complementary DNA. Thus, the alkylating derivative of the T7 DNA restriction fragment retains its capacity to hybridise with T7 DNA, however, it does not interact with the noncomplementary DNA from chick erythrocytes, it was established that the alkylating derivative of DNA fragment efficiently alkylates only the complementary DNA.

Alkylating Agents↗
PubMed1995

DNA-strand breaks induced by dimethylarsinic acid, a metabolite of inorganic arsenics, are strongly enhanced by superoxide anion radicals.

We previously reported that dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induced DNA single-strand breaks (ssb) both in vivo and in cultured alveolar type II (L-132) cells in vitro, possibly via the production of dimethylarsenic peroxyl radicals. Here, the interaction of superoxide anion radicals (O2-) in the induction of ssb in L-132 cells was investigated using paraquat, an O2(-)-producing agent. A significant enhancement of ssb formation was observed in the DMAA-exposed cells when coexposed to paraquat. This enhancement occurred even when post-exposed to DMAA after washing, suggesting that the DMAA exposure caused some modification of DNA such as DNA-adducts, which was recognized by active oxygens to form ssb. An experiment with UV-irradiation, which was likely to induce ssb at the modified region, supported the possibility of DNA modification by DMAA exposure. An ESR study indicated that O2- produced by paraquat in DMAA-exposed cells was more consumed than in non-exposed cells, assumingly through the reaction with the dimethylarsenic-modified region of DNA. The species of active oxygens were estimated by using diethyldithiocarbamate, aminotriazole, diethylmaleate, hydrogen peroxide (H2O2), gamma-irradiation and ethanol. O2- but neither H2O2 nor hydroxyl radicals was very likely to contribute to the ssb-enhancing action of paraquat.

Cacodylic Acid↗
PubMed1998

Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects.

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 microM), while alpha-tocopherol and the iron chelator alpha-phenanthroline had no effect and beta-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

Animals↗
PubMed1995

In vivo DNA adduct formation by bisphenol A.

We have previously shown that bisphenol A (BPA) is oxidized to bisphenol-o-quinone in the presence of activation system and that the chemical reaction of DNA or deoxyguanosine 3'-monophosphate (dGMP) with bisphenol-o-quinone produces adducts. In the present study, using the 32P-postlabeling technique, we have investigated the in vivo DNA adduct formation by BPA by examining covalent modification in DNA. Administration of a single or multiple dose of 200 mg/kg of BPA to CD1 male rats produced two major and several minor adducts in liver DNA. The two major in vivo adducts matched the adduct profile of DNA or dGMP-bisphenol-o-quinone. To determine how BPA may be converted to DNA-binding metabolites, adducts were examined after incubation of DNA with BPA in the presence of a microsomal activation system. The in vitro incubation of BPA with DNA in the presence of a microsomal activation system revealed one major adduct and several minor adducts. The formation of adducts in DNA by BPA in the presence of a microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. Adduct formation in DNA when cumene hydroperoxide or NADPH was used as a cofactor showed adducts with similar chromatographic mobilities as those from the reaction of dGMP-bisphenol-o-quinone. These data demonstrate that BPA is capable of binding covalently to DNA. DNA binding can be inhibited by the inhibitors of cytochrome P450. One of the DNA-binding metabolite(s) both in vitro and in vivo may be bisphenol-o-quinone. Covalent modifications in DNA by in vivo exposure of BPA may be a factor in the induction of hepatotoxicity.

Animals↗
PubMedDate not supplied

Role of endogenous oxidative DNA damage in carcinogenesis: what can we learn from repair-deficient mice?

Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) are observed in all types of cells, most probably due to a continuous generation of reactive oxygen species (ROS) in the cellular oxygen metabolism, and it has long been suspected that they might play an important role in the initiation of carcinogenesis. Experimental evidence for this assumption can be obtained by studying the effects of a modulation of the steady-state levels, either by in- or decreasing the generation of oxidative DNA damage, on spontaneous mutation rates and cancer incidence. However, clear answers have not yet been obtained by these strategies. It is still doubtful whether an efficient reduction of the in vivo steady-state levels can be achieved by application of antioxidants, and effects observed under oxidative stress conditions (i.e. increased oxidative DNA damage) are inconclusive due to the pronounced epigenetic effects of ROS on signal transduction and gene expression (tumor promotion). In addition, the reliable quantification of the basal levels of oxidative DNA modifications is still a major problem. Recently, the generation of mice deficient in the repair 8-oxoG (ogg1-/- mice) has opened the door for an alternative approach. Results obtained so far indicate that an increase by less than five 8-oxoG residues per 106 bp in the liver of the knockout animals is associated with a two- to threefold higher spontaneous mutation frequency in transgenic genes. However, the increase in the ogg1-/- mice of the steady-state level of 8-oxoG and the spontaneous mutation frequency was only observed in the liver and apparently too small to enhance the spontaneous cancer incidence significantly. The limited effect seems to be due to a back-up repair system for 8-oxoG in the ogg1-/- mice, and it can be expected that the inactivation of this pathway in double-knockout mice will lead to higher effects and a better assessment of the risk associated with endogenous oxidative DNA damage.

Animals↗