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Nucleic Acids Research Feb 19952-Hydroperoxytetrahydrofuran (THF-OOH) can be employed to sequence cytosine (C) and to probe for non-canonical DNA structures involving C. Using 32P-labeled oligomers...
2-Hydroperoxytetrahydrofuran (THF-OOH) can be employed to sequence cytosine (C) and to probe for non-canonical DNA structures involving C. Using 32P-labeled oligomers and a DNA restriction fragment, it is demonstrated that THF-OOH has a strong preference for Cs in single-stranded (s-s) DNA regions, and in bulges, loops and mismatches. The reactivity of C is diminished below pH 6.0, but is not affected by substitution of 5-methylcytosine. To demonstrate the utility of the reagent, it is directly compared to methoxylamine and chloroacetaldehyde, two other reagents commonly used to chemically probe C residues in non-Watson-Crick DNA structures.
Topics: 5-Methylcytosine; Acetaldehyde; Base Sequence; Cytosine; DNA; DNA, Single-Stranded; Furans; Hot Temperature; Hydrogen-Ion Concentration; Hydroxylamines; Indicators and Reagents; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; Sequence Analysis, DNA
PubMed: 7899093
DOI: 10.1093/nar/23.4.713 -
The Biochemical Journal Sep 19801. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2....
1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).
Topics: Antithrombin III; Arginine; Electrophoresis, Polyacrylamide Gel; Humans; Hydrolysis; Hydroxylamines; Macromolecular Substances; Models, Chemical; Molecular Weight; Protein Binding; Thrombin
PubMed: 7213342
DOI: 10.1042/bj1890481 -
Organic Letters Jul 2013Generation of the 5-(2'-deoxyuridinyl)methyl radical (6) was reexamined. Trapping by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl confirms that 6 is generated. However,...
Generation of the 5-(2'-deoxyuridinyl)methyl radical (6) was reexamined. Trapping by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl confirms that 6 is generated. However, trapping by methoxyamine reveals that the respective carbocation (10) is also produced. Examining the effects of these traps on products in DNA reveals that the carbocation and not 6 yields interstrand cross-links. Cross-link formation from the carbocation is consistent with DFT calculations that predict that addition at the N1 position of dA is essentially barrierless.
Topics: Cross-Linking Reagents; Cyclic N-Oxides; Deoxyuridine; Molecular Structure; Pyrimidines; Spin Labels
PubMed: 23822109
DOI: 10.1021/ol401472m -
Nucleic Acids Research Aug 1992The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and...
The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.
Topics: Bacteriophages; Base Sequence; Cytosine; DNA Replication; DNA, Viral; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Escherichia coli; Molecular Sequence Data; Mutagenesis; Nucleic Acid Conformation; Nucleic Acid Synthesis Inhibitors; Oligodeoxyribonucleotides; Rec A Recombinases; Sulfites; Templates, Genetic
PubMed: 1508715
DOI: 10.1093/nar/20.16.4213 -
Nucleic Acids Research Jul 1977Cytosine residues in 32P-labeled E. coli tRNA Leu 1 were modified by treatment of the tRNA with the semicarbazide-bisulfite reagents [Hayatsu, H. (1976) Biochemistry 15,...
Cytosine residues in 32P-labeled E. coli tRNA Leu 1 were modified by treatment of the tRNA with the semicarbazide-bisulfite reagents [Hayatsu, H. (1976) Biochemistry 15, 2677-2682]. Analysis of the modification sites showed that only four cytidine residues, i.e. C35, C53, C85 and C86, reacted. They were identical with the cytidines of this tRNA accessible to methoxyamine [Chang, S. E. and Ish-Horowicz, D. (1974) J. Mol. Biol. 84, 375-388] and the accessibility was consistent with the conformational features recognized for tRNA in general. The rapidity and the simple nature of this modification demonstrate that the semicarbazide-bisulfite reaction is a useful tool in studying conformations of polynucleotides.
Topics: Base Sequence; Cytosine; Escherichia coli; Leucine; Nucleic Acid Conformation; Oligoribonucleotides; RNA, Transfer; Ribonuclease T1; Ribonucleotides; Semicarbazides; Sulfites
PubMed: 409997
DOI: 10.1093/nar/4.7.2283 -
Proceedings of the National Academy of... May 1989We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the...
We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.
Topics: Adenine; Animals; Cell Line; Cricetinae; DNA; DNA Damage; DNA Probes; DNA Repair; Deoxyribonucleases, Type II Site-Specific; Dimethyl Sulfoxide; Electrophoresis, Agar Gel; Exons; Guanine; Hot Temperature; Hydrolysis; Nucleic Acid Hybridization; RNA Probes; Sulfuric Acid Esters; Tetrahydrofolate Dehydrogenase; Transcription, Genetic
PubMed: 2785688
DOI: 10.1073/pnas.86.9.3050 -
Journal of the American Society For... Nov 2009Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione...
Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: a combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study.
Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species.
Topics: Electron Spin Resonance Spectroscopy; Glutathione; Hydroxylamine; Hydroxylamines; Molecular Structure; Nitroso Compounds; Oxidation-Reduction; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 19744863
DOI: 10.1016/j.jasms.2009.07.018 -
The Journal of Biological Chemistry Oct 1989Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to... (Comparative Study)
Comparative Study
Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.
Topics: DNA; DNA Glycosylases; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Endodeoxyribonucleases; Hydrogen-Ion Concentration; Hydroxylamines; Kinetics; Micrococcus; Multienzyme Complexes; N-Glycosyl Hydrolases; Osmolar Concentration; Plasmids; Pyrimidine Dimers; Sodium Chloride; Ultraviolet Rays
PubMed: 2477371
DOI: No ID Found -
Journal of the American Chemical Society Jan 2013We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the...
On the formation and properties of interstrand DNA-DNA cross-links forged by reaction of an abasic site with the opposing guanine residue of 5'-CAp sequences in duplex DNA.
We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5'-CAp sequence under conditions of reductive amination (NaCNBH(3), pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions, and the cross-link, once formed, was stable to a variety of workup conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LC-MS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N(2)-amino group of the opposing guanine residue in 5'-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH(3)/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N(2)-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH(3).
Topics: 5' Untranslated Regions; Aldehydes; Chromatography, Liquid; Cross-Linking Reagents; DNA; Guanine; Models, Molecular; Molecular Structure; Tandem Mass Spectrometry
PubMed: 23215239
DOI: 10.1021/ja308119q -
The Biochemical Journal Nov 1997In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved...
In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.
Topics: Amino Acid Substitution; Animals; Apoenzymes; Arginine; Binding Sites; Carboxylic Acids; Chromatography, Gel; Circular Dichroism; Coenzymes; Cytosol; Enzyme Stability; Glycine Hydroxymethyltransferase; Liver; Mutagenesis, Site-Directed; Recombinant Proteins; Sheep; Spectrophotometry, Ultraviolet
PubMed: 9581569
DOI: 10.1042/bj3270877