The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine

Wyatt, G.R.; Cohen, Seymour S.

doi:10.1042/bj0550774

Cited by 574 publications

(136 citation statements)

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“…Synthetic polydeoxynucleotides were determined by their optical absorption using the extinction coefficient ~2 6~ n,cL= 6.7 x iO3cm-1M-l for poly d(A-T) and 6.5 x 103cm-lM-l for poly(dG) * poly(dC) [24]. To determine the base composition the nucleic acids were hydrolyzed in formic acid [25] and the bases separated by twodimensional thin-layer chromatography on MN 300 cellulose (Macherey, Nagel and Co., Duren) with methanol-HC1-water (65 : 1'7 : 18) and n-butanol (saturated with water)-NI-I, (100: 1) as solvents. After elution of the ultraviolet quenching spots the concentrations were determined spectrophotometrically.…”

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confidence: 99%

Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA*

Behr

Honikel

Hartmann

1969

European Journal of Biochemistry

145

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Formation of a complex between chromomycin and DNA has been shown to depend on the guanine content and on base pairing of the polydeoxynucleotides. A limit of association is reached when one chromomycin molecule is bound per four nucleotide base pairs. The rate of complex formation is independent of the size of the sugar side chains of the antibiotic and of the nature and base composition of DNA. I n contrast the rate of complex dissociation increases with a decrease in size of the sugar side chains. The inhibition by chromomycin and its derivatives of DNA and RNA polymerase reactions exhibits similar features. Probably the dissociation rate of the complex determines the inhibitory activity. Chromomycin by complex formation protects guanine rich segments of DNA very efficiently against the attack of nucleases. After enzymatic hydrolysis chromomycin containing base paired oligonucleotide fragments remain in the reaction mixture and can be isolated by electrophoresis. The antibiotic is not covalently bound to these fragments and can be removed by thorough extraction with ether.Low concentrations of the antibiotic chromomycin Id and of closely related compounds such as olivomycin and mithramycin have been shown to inhibit selectively the DNA directed cellular and enzymatic RNA synthesis [l-91. The DNA polymerase reaction is also retarded [6]. The following RO

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confidence: 99%

Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA*

Behr

Honikel

Hartmann

1969

European Journal of Biochemistry

145

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“…The percentage of these elements was highest in the nucleic acid of T2, intermediate in that of T4, and lowest in that of the T6 virus. The molar ratios of N/P varied between 3.72 and 3.86, and were slightly higher than the value, 3.69, calculated from the molar proportions of the nucleic acid bases reported earlier (1). Since all the nucleic acid preparations contained small quantities of protein (0.9 to 1.4 per cent), it is possible that the elevated value of the N/P ratio might be due to this component.…”

Section: Properties Of the Viral Nucleic Acids-mentioning

confidence: 59%

“…Since each nucleic acid (1,22) contains essentially the same quantity of hydroxymethylcytosine (0.163 to 0.168 moles per 1 mole of phosphorus), it is apparent that the molar ratio of glucose to hydroxymethyl-cytosine is in each instance different (0.81, 1.08, and 1.71, respectively). It is obvious, therefore, that in the T2 nucleic acid the number of glucose molecules is less than the number of hydroxymethylcytosine molecules.…”

Section: Properties Of the Viral Nucleic Acids-mentioning

confidence: 99%

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THE NUCLEIC ACIDS OF T2, T4, AND T6 BACTERIOPHAGES

Jesaitis

1957

The Journal of Experimental Medicine

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Some years ago it was shown by W y a t t and Cohen (1) that the nucleic acids of the three coil-dysentery phages, T~, T4, and T6, contain nearly identical quantities of the same purine and pyrimidine bases and that they differ from the deoxyribonucleic acids derived from other sources in that they contained hydroxymethylcytosine instead of cytosine or its 5-methyl derivative. It was subsequently found in this laboratory that a hexose was present in the T, phage (2). This sugar was identified as glucose and was shown to be a constituent of the viral nucleic acid (3). Sinsheimer (4) and Volkin (5) have shown that the T, and Te phages also contain glucose and that this saccharide can, in part, be isolated as a glucoside of hydroxymethylcytidylic acid from enzymatic hydrolysates of T~ and T, nucleic acids. I t was recently reported that the nucleic acids of the three even numbered T phages contained varying amounts of glucose (6-8).I n the present communication the chemical properties of the nucleic acids of the wild type strains (r +) of T~, T,, and T6 phages will be described and the differences in their chemical composition will be discussed. It will be shown that the three nucleic acids differ not only in their content of glucose but also in that they contain chemically different hydroxymethylcytosine mononucleotides. Materials and MethodsBazteriopkages.--The wild type (r +) strains of T~, T,, and T6 phages used in this study were originally obtained from Dr. Mark H. Adams of New York University. They were maintained by occasional transfer on E. coU B in nutrient broth. Mass cultures of T2 and T( phage were prepared by infecting E. coli B grown in a glucose-phosphate buffer medium (9).In order to propagate the T6 phage successfully it was necessary to add 0.1 per cent Difco nutrient broth shortly before infecting the culture with virus. The phages themselves were isolated and purified as previously described (9).Analyt/ca/MeJhods.--The nitrogen and phosphorus content of the materials studied was determined colorimetrically by the procedures of Koch and McMeekin (10) and of Allen (11). Protein was determined with Folin reagent (12) using crystalline bovine albumen as a standard. The hexose content of the nucleic acids and of their degradation products was determined colorimetrically by means of the anthrone reaction as follows (13) : 1 ml. portions of an aqueous solution of ,mknown substance were placed in test tubes of 15 ram. diameter. To each was added 0.2 ml. of a 2.5 per cent solution of anthrone in ethyl acetate followed by 2.5 mh of concentrated sulfuric acid. The reagents were mixed and the 233

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“…At present, all known TET enzymes (TET1-3) are Fe(II)-and 2-ketoglutarate-dependent oxidases, which efficiently convert 5mC to 5hmC both in vitro and in vivo [3]. Aside from functioning as the epigenetic regulator in the mammals, 5hmC was first identified in bacteriophage genomes as a strategy to evade bacterial restriction endonucleases [4]. Moreover, the hydroxymethylation is catalyzed by dCMP hydroxymethylase (CH), which transfers methylene group from methylenetetrahydrofolate (CH 2 THF) to C 5 of dCMP and then uses water molecule to hydrate the methylene group to form hydroxymethyl-dCMP (HmdCMP), one precursor for phage DNA replication [5].…”

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Evidence from 18O feeding studies for hydroxyl group donor in the reaction catalyzed by cytidylate hydroxymethylase MilA

et al. 2013

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5-Hydroxymethylcytosine (5hmC) was present in T-even phage and mammalian DNA. 5hmC in phage is formed by hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) by deoxycytidylate hydroxymethylase (CH), which uses the solvent water as the hydroxyl group donor. By contrast, 5hmC is formed in mammal zygotes by the oxidation of 5-methylcytosine (5mC). 5hmC was also present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA. However, the catalytic mechanism remains unknown. In the present study, we purified His-tagged MilA and fed its in vitro reaction with H 2 18 O. The LC-MS analysis of the product revealed that 18 O was incorporated into the hydroxymethylated CMP (HmCMP), and the secondary MS result of 18 O-labeled HmCMP indicated that 18 O was incorporated into the cytosine of HmCMP. The results demonstrate that MilA uses solvent water as the hydroxyl group donor like CH. Moreover, Thr102 of MilA was predicted as potential critical amino acid anchoring one molecule of water for hydroxylation. Finally, organizational context comparison in microbial genomes reveals that six homologous ORFs originally annotated as putative thymidylate synthase (TS) are more likely to be CMP hydroxymethylase. Since the discovery of the "sixth base", 5-hydroxymethylated cytosine (5hmC) in mammalian DNA, the enzymology behind the formation of this new base has drawn world-wide attention recently. Such an attention stems from Rao and coworkers' discovery via bioinformatics analysis that 5hmC is formed post-replicatively by Tet1-catalyzed oxidation of 5-methylated cytosine (5mC) [1]. 5hmC in DNA could be further oxidized by Tet3 dioxygenases in vitro to 5-carboxylcytosine that is specifically recognized and excised by thymine-DNA glycosylase (TDG), and therefore Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization [2]. At present, all known TET enzymes (TET1-3) are Fe(II)-and 2-ketoglutarate-dependent oxidases, which efficiently convert 5mC to 5hmC both in vitro and in vivo [3]. Aside from functioning as the epigenetic regulator in the mammals, 5hmC was first identified in bacteriophage genomes as a strategy to evade bacterial restriction endonucleases [4]. Moreover, the hydroxymethylation is catalyzed by dCMP hydroxymethylase (CH), which transfers methylene group from methylenetetrahydrofolate (CH 2 THF) to C 5 of dCMP and then uses water molecule to hydrate the methylene group to form hydroxymethyl-dCMP (HmdCMP), one precursor for phage DNA replication [5]. 5-hydroxymethylcytosineBesides the occurrence of 5hmC on the chromosomal

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The bases of the nucleic acids of some bacterial and animal viruses: the occurrence of 5-hydroxymethylcytosine

Cited by 574 publications

References 18 publications

Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA*

Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA*

THE NUCLEIC ACIDS OF T2, T4, AND T6 BACTERIOPHAGES

Evidence from 18O feeding studies for hydroxyl group donor in the reaction catalyzed by cytidylate hydroxymethylase MilA

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