Products distribution in the oxidation of thymine with hydroxyl radicals

In the oxidation of thymine with hydroxyl radical generated from L-ascorbic acid/copper(II) ion/O system, four reaction products, thymine glycol(TG), N-formyl-N'-pyruvylurea(FPU), 5-hydroxymethyluracil(HMU) and 5-hydroxy-5-methyl barbituric acid(HMBA) were obtained. A reaction scheme was proposed to explain the products distribution observed.     

Methylation of thymine and uracil with free methyl cations formed due to beta-decay of tritiated methane: possible implication in mutagenesis and carcinogenesis

Exposure of solid thymine and uracil at room temperature to free methyl cations, produced due to beta-decay of tritiated methane, resulted in formation of their 1-, O2-, 3-, O4-, and 6-methyl derivatives. In addition, uracil formed a 5-methyl derivative (thymine); tritium-containing thymine and uracil were also detected. Both thymine and uracil formed predominantly unidentified products which resulted presumably from their oligomerization. Incubation at -195 degrees C did not markedly change the pattern of reaction products. Aqueous-ammonia solutions of these pyrimidines formed methylated derivatives and considerable amounts of methanol and tritiated water. The possible implication of these reactions in mutagenic and carcinogenic effects of tritium-substituted hydrocarbons is discussed.     

Hydroxymethyluracil modifications enhance the flexibility and hydrophilicity of double-stranded DNA

Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores, short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications for recognition and processing of such modifications by regulatory DNA-binding proteins.     

Thymine 7-hydroxylase from Neurospora crassa. Substrate specificity studies.

A partially purified preparation of thymine 7-hydroxylase (thymine, 2-oxoglutarate : oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) from Neurospora crassa was incubated with a number of pyrimidines chemically related to tyymine. 1. Pyrimidines with oxygen or sulfur substituents on atoms Nos. 2 and 4 as well as an alkyl group on atom Nos. 1 or 5 were substrates. 2. Km values were determined for 1-methyluracil, 1-ethyluracil, thymine, 6-azathymine, 1-methylthymine, 1-ethylthymine, 5-formyluracil and 5-hydroxymethyluracil. 3. Uracil was identified as one of the metabolites after incubation with 1-methyluracil. The one-carbon metabolite has not been characterized. 4. Several pyrimidines with polar groups on atoms Nos. 2 and 4 were inhibitory. 5. Addition of 1-methyluracil, 1-methylthymine, 1-ethylthymine or 5-hydroxymethyluracil to incubations with thymine and 2-oxo[1-14C1]glutarate did not result in additional formation of 14CO2, indicating that the same enzyme acts on the different compounds. It has previously been found (Bankel, L., Holme, E., Lindstedt, G. and Lindstedt, S. (1972) FEBS Lett. 21, 135-138) that a mutant strain of N. crassa which is devoid of thymine 7-hydroxylase activity also lacks ability to perform the coupled oxygenation of 2-oxoglutarate and 1-methyluracil, 5-hydroxymethyluracil and 5-formyluracil, respectively. It is concluded that one and the same oxygenase is responsible for the activities studied.     

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.     

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.     

Determination of intra- and intermolecular tritium isotope effects in the reaction of thymine 7-hydroxylase

Different [7-3H]thymine preparations have been used to determine the inter- and intramolecular isotope effects of the 2-oxoglutarate-dependent thymine hydroxylation, catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.6). Specific activity ratios of products, viz., 3H2O and 5-hydroxymethyluracil, and remaining substrate to initial substrate have been determined. The influence on these ratios of intra- and intermolecular isotope effects at different degrees of tritium substitution has been analyzed. An intramolecular isotope effect with a kH/kT of about 6.5 has been found. No intermolecular isotope effect of TV/K could be detected when oxygen concentration was varied from 0.4 to 0.01 mM. This agrees with a mechanism in which 2-oxoglutarate is irreversibly changed before the bond-breaking in thymine takes place.     

Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA

A specific and efficient method is presented for the conversion of 2′-deoxyuridine to thymidine via formation and reduction of the intermediate 5-hydroxymethyl derivative. The method has been used to generate both thymidine and 5-methyl-2′-deoxycytidine containing the stable isotopes ²H, ¹³C and ¹⁵N. Oligodeoxyribonucleotides have been constructed with these mass-tagged bases to investigate sequence-selectivity in hydroxyl radical reactions of pyrimidine methyl groups monitored by mass spectrometry. Studying the reactivity of 5-methylcytosine (5mC) is difficult as the reaction products can deaminate to the corresponding thymine derivatives, making the origin of the reaction products ambiguous. The method reported here can distinguish products derived from 5mC and thymine as well as investigate differences in reactivity for either base in different sequence contexts. The efficiency of formation of 5-hydroxymethyluracil from thymine is observed to be similar in magnitude in two different sequence contexts and when present in a mispair with guanine. The oxidation of 5mC proceeds slightly more efficiently than that of thymine and generates both 5-hydroxymethylcytosine and 5-formylcytosine but not the deaminated products. Thymine glycol is generated by both thymine and 5mC, although with reduced efficiency for 5mC. The method presented here should be widely applicable, enabling the examination of the reactivity of selected bases in DNA.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Catalysis of three sequential dioxygenase reactions by thymine 7-hydroxylase

A purification scheme has been developed for an enzyme, thymine 7-hydroxylase, which appears to catalyze three sequential dioxygenase reactions, i.e., thymine → 5-hydroxymethyluracil → formyluracil → uracil-5-carboxylic acid. The enzyme was purified 1,300-fold from Neurospora crassa and had specific activities of approximately 1200, 600, and 250 U/mg for the respective reactions. Evidence that a single protein catalyzes the three reactions includes: the parallel purification of the three activities throughout the purification scheme, the inhibition of each reaction by the substrates of the other two, the inhibition of the three reactions by uracil, the parallel loss of the three activities upon heat denaturation, and considerations of a mechanism which suggest that a single active site may be involved.     

Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1

Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.     

Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.     

Enzymatic excision of radiation-induced lesions from DNA model compounds.

Dinucleoside monophosphates in which the 5' nucleoside contained a radiation-modified base were tested as substrates to bovine spleen phosphodiesterase (SPD) and snake venom phosphodiesterase. The radiation-modified bases included thymine glycols, 5-hydroxymethyluracil, 8-hydroxyguanine, and a formamido remnant of thymine. The lesions had widely different effects on diesterase action, varying from little inhibition, as in the case of digestion of dT*pA by SPD, where T* is the hydroxymethyluracil modification, to severe inhibition, as in the case of digestion of dG*pC by SPD, where G* is the 8-hydroxyguanine modification.     

Mutants of Bacillus subtilis Bacteriophage φe Defective in dTTP-dUTP Nucleotidohydrolase

Mutants of bacteriophage phie inducing only 1 to 5% of wild-type levels of dTTP-dUTP nucleotidohydrolase give normal bursts of viable progeny phage whose DNA contains 5 to 10% thymine (but no uracil) in place of 5-hydroxymethyluracil. The relative heat lability of one phage mutant enzyme solubilized from the membrane fraction of infected cells suggests that a phie gene codes for the induced dTTPase-dUTPase.     

Substitution of nucleoside triphosphates for ascorbate in the thymine 7-hydroxylase reaction of Rhodotorula glutinis

Nucleoside di- and triphosphates substituted for ascorbate in the thymine 7-hydroxylase reaction in studies carried out with purified preparations from Rhodotorula glutinis. The stimulations brought about by ascorbate and ATP were found not to be additive. Studies with analogues of ATP indicated that hydrolysis may not need to occur in order for the nucleotide effect to be expressed. The stoichiometry of the production of 5-hydroxymethyluracil and CO2 was not changed by the substitution of ATP for ascorbate. The 7-hydroxylase was found to have considerable thermal stability, and inactivation at 98 degrees C resulted in a parallel loss of the activities effected by ascorbate and ATP. This and the retention of the nucleotide effect upon purification suggest the effect is not mediated through another protein co-purified with the 7-hydroxylase.     

A kinetic study of thymine 7-hydroxylase from neurospora crassa.

The steady-state kinetics of thymine 7-hydroxylase (thymine, 2-oxoglutarate dioxygenase, EC 1.14.11.6) has been investigated. Initial velocity plots were all found to be linear and intersecting. Variation in concentration of two of the substrates, when the third substrate was at a constant high or low concentration, gave initial velocity plots that conform to an ordered sequential mechanism, where thymine is the second substrate to add. With 5-carboxyuracil, which is the end product in the sequential oxygenation of thymine, a competitive inhibition pattern was observed when 2-ketoglutarate was the variable substrate. When either thymine or oxygen was the variable substrate a noncompetitive inhibition pattern was obtained. When either 2-ketoglutarate or thymine was the variable substrate the inhibition patterns observed with bicarbonate were noncompetitive. With succinate noncompetitive inhibition patterns with hyperbolic intercept replots were obtained. These results are consistent with an ordered sequential kinetic mechanism, where 2-ketoglutarate is added first, followed by thymine and oxygen, and the products are released in the order: bicarbonate, succinate, and 5-hydroxymethyluracil. The order of the two last mentioned products, however, is changed in the presence of succinate.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.     

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.