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J R Mehta

Publications and source records attributed to J R Mehta.

30 records · Page 2Linked to original sources
PubMed1986

Formation of O6-ethylthioethylguanine in DNA by reaction with the sulfur mustard, chloroethyl sulfide, and its apparent lack of repair by O6-alkylguanine-DNA alkyltransferase.

The reactions of the monofunctional sulfur mustard, chloroethyl ethyl sulfide, with calf thymus DNA have been studied in vitro. In addition to extensive alkylation of the 7 position of guanine and the 3 position of adenine, approximately 0.1% of the total alkylation is on the O6 position of guanine. Since chloroethyl ethyl sulfide is a model for S-adenosylmethionine and for the reactive intermediates generated from glutathione by certain environmental agents, this result establishes a route by which these compounds could be mutagenic. A DNA substrate modified for repair studies has been produced by incubating calf thymus DNA with [14C]chloroethyl-labelled chloroethyl ethyl sulfide. This DNA contains 6 nmol/mg of alkylated products including 6 pmol/mg O6-ethylthioethylguanine. Incubation of this substrate with up to a 20-fold excess of mammalian O6-alkylguanine-DNA alkyltransferase has indicated that O6-ethylthioethylguanine is either not a substrate or a poor one for this DNA repair system.

Adenine↗
PubMed1986

Prevention of 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane cross-link formation in DNA by rat liver O6-alkylguanine-DNA alkyltransferase.

Previous studies have shown that the haloethylnitrosoureas introduce the cross-link 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane into DNA. This structure is evidently formed by the following sequence of events: an initial attack of a haloethyl group on the O6 position of guanine, formation of the reactive intermediate, 1,O6-ethanoguanine, and reaction of this intermediate with deoxycytidine in the opposite DNA strand. To investigate the role of O6-alkylguanine-DNA alkyltransferase in preventing the formation of this cross-link, a DNA substrate containing O6-fluoroethylguanine has been prepared by reacting DNA with N-2-fluoroethyl-N'-cyclohexyl-N-nitrosourea. The O6-fluoroethylguanine content of this substrate decreases when it is incubated at 37 degrees C and pH 7.8 in the absence of repair factors because of the chemical instability of O6-fluoroethylguanine; however, this loss is accelerated by the addition of rat liver O6-alkylguanine-DNA alkyltransferase, indicating that this repair factor recognizes and repairs O6-fluoroethylguanine in DNA; furthermore, by using [chloroethyl-14C]N-chloroethyl-N'-cyclohexyl-N-nitrosourea, it can be shown directly that the addition of rat liver O6-alkylguanine-DNA alkyltransferase prevents 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane formation. These studies link the presence of repair activity to the prevention of a specific cytotoxic lesion in DNA.

Alkylation↗
PubMed1984

Formation of O6-ethylthioethyldeoxyguanosine from the reaction of chloroethyl ethyl sulfide with deoxyguanosine.

O6-Ethylthioethyldeoxyguanosine has been synthesized from 6-chloro-3',5'-di-O-acetyldeoxyguanosine and characterized by UV, fluorescence, and mass spectrometry. High-pressure liquid chromatography studies have shown that this modified nucleoside is formed when the one-armed sulfur mustard, chloroethyl ethyl sulfide, reacts with deoxyguanosine. This result supports the hypothesis that the mutagenic effects of the sulfur mustards are caused in part by substitution of the O6-position of deoxyguanosine.

Chromatography, High Pressure Liquid↗
PubMed1983

Properties of the chromatin repair activity against O6-ethylguanine lesions in DNA. Mechanism of the reaction.

Chromatin proteins from rat liver contain a repair activity that removes O6-ethylguanine from ethylnitrosourea-treated DNA. This activity does not depend on divalent cations and works in the presence of EDTA, but does depend on the presence of free thiol groups. Thus, it is destroyed by N-ethylmaleimide and is protected by dithiothreitol. The repair activity on single-stranded DNA is only 20% of what it is on double-stranded DNA; its half-life at 35 degrees C is 55 min, but DNA, ethylated or not, affords some protection. The repair reaction is a transethylation from O6-ethylguanine in DNA onto two different cysteine residues contained in acceptor proteins. The reaction can be followed by monitoring the appearance of ethylated proteins or by disappearance of O6-ethylguanine from DNA.

Animals↗
PubMed1982

Trapping of DNA-reactive metabolites of therapeutic or carcinogenic agents by carbon-14-labeled synthetic polynucleotides.

Many substances which do not react with DNA directly are metabolized into important DNA-modifying intermediates. We have devised a method for trapping these intermediates with 14C-labeled nucleosides contained in a synthetic polynucleotide. The polynucleotide structure protects the labeled nucleoside from metabolism; thus, it is unaltered when the polymer is incubated with a drug-metabolizing system. However, when the polymer is incubated with this system and a compound which can be metabolized into a reactive species, these intermediates are trapped by the 14C-labeled nucleoside and subsequently are detected as new peaks of radioactivity in a digest of the labeled polynucleotide. This system has been used to detect reactive intermediates of cyclophosphamide generated by a liver homogenate.

Animals↗
PubMed1981

Repair of O6-ethylguanine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group to an acceptor protein.

Incubation of O6-[3H]ethylguanine-containing DNA with a rat liver chromatin fraction resulted in a decrease in the O6-ethylguanine content of the DNA. Analysis of the products of this reaction showed that the ethyl group had been transferred from the O6-ethylguanine to a protein acceptor. When the incubation mixture was separated on a cesium chloride gradient, the radioactivity removed from O6-ethylguanine appeared in a low-density band. This material has been isolated and subjected to trypsin digestion and high-pressure liquid chromatography analysis; it was sensitive to trypsin and the digest contained new high-pressure liquid chromatography peaks characteristic of oligopeptides. Radioactive peaks from the trypsin digestion have been digested further to the amino acid level and have been shown to contain S-[3H]ethylcysteine. Thus, we conclude that the repair activity in rat liver chromatin removes the ethyl group from O6-ethylguanine and transfers it to a cysteine moiety contained in an acceptor protein.

Animals↗
PubMed1980

Synthesis of 8-14C-labeled O6-methyldeoxyguanosine and its deoxynucleotide copolymers.

To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, we have synthesized 8-14C-labeled O6-methyldeoxyguanosine triphosphate and investigated the kinetics of its incorporation into the synthetic copolymers poly(dC'm6dG) and poly(dT,m6dG). Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5'-monophosphate with carrot phosphotransferase and then to the 5'-triphosphate via the phosphorimidazolidate formed by the action of N,N'-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was, however, higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was found to be pH dependent, and the half-life has been measured at four different pH values.

DNA Polymerase I↗
PubMed1980

Alkylation of guanosine and deoxyguanosine by phosphoramide mustard.

Phosphoramide mustard, an active metabolite of cyclophosphamide, has been reacted separately with guanosine and deoxyguanosine in aqueous solution at pH 7.4. The major adduct which was formed in each case has been isolated by reverse-phase high-pressure liquid chromatography. The structure of the major adduct, as determined by a combination of ultraviolet and field desorption mass spectrometry, is that of phosphoramide mustard, one arm of which has reacted with guanosine or deoxyguanosine in position 7. These adducts are much less stable than was 7-methylguanosine, and they decompose with a half-life of 1.3 hr at 37 degrees and pH 7.4. This instability may contribute to the action of phosphoramide mustard at a molecular level.

Alkylation↗
PubMed1978

Synthesis and properties of O6-methyldeoxyguanylic acid and its copolymers with deoxycytidylic acid.

This paper describes the synthesis of O6-methyldeoxyguanosine triphosphate (m6dGTP) and its copolymerization to high molecular weight polymer with deoxycytidylic acid. The monomer, m6dGTP, was synthesized from deoxyguanosine first protected by acetylation of the sugar hydroxyls, and then chlorinated in the 6-position with POCl3. The product, 6-chloro-3',5'-di-O-acetyl deoxyguanosine, was converted to O6-methyldeoxyguanosine with sodium methoxide and phosphorylated in the 5' position with carrot phosphotransferase. Monophosphate was converted chemically to the triphosphate and copolymerized with dCTP by terminal deoxynucleotidyl transferase. The resulting template, which contained O6-methylguanine, was tested for its ability to direct RNA synthesis by bacterial RNA polymerase. The presence of O6-methylguanine was shown to lead to the misincorporation of UMP in the product polymer, thus strengthening the hypothesis that O6-methylguanine is a promutagenic base.

Deoxycytidine Monophosphate↗
PubMed1976

Synthesis and properties of poly(O6-methylguanylic acid) and poly(O6-ethylguanylic acid).

The nucleotide analogues, O6-methyl- and O6-ethylguanosine diphosphate, have been synthesized and polymerized to high-molecular-weight homopolymers with polynucleotide phosphorylase. The ultraviolet spectra of these polymers show marked hypochromicity, which suggests that they possess considerable secondary structures. Graphs of optical denisty vs. temperature in 0.15 M NaC1 indicate that cooperative melting occurs for both polymers, and that the secondary structure of poly(O6-methylguanosine monophosphate) is somewhat more stable than that of poly (O6-ethyl-guanosine monophosphate). Mixing experiments show that these analogue polymers no longer form helical structures with poly(C), nor do thye form helices with poly(U). We would conclude from these data that environmental mutagens and carcinogens which react at the O6 position of guanine not only disrupt normal base-pairing relationships, but may also affect the secondary structure of nucleic acids.

Chromatography, Paper↗