5-formyluracil and 5-hydroxymethyluracil

We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.

Topics: Base Sequence; DNA; Models, Molecular; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Pentoxyl; Thymine; Uracil

TET proteins play a vital role in active DNA demethylation in mammals and thus have important functions in many essential cellular processes. The chemistry for the conversion of 5mC to 5hmC, 5fC and 5caC catalysed by TET proteins is similar to that of T to 5hmU, 5fU and 5caU catalysed by thymine-7-hydroxylase (T7H) in the nucleotide anabolism in fungi. Here, we report the crystal structures and biochemical properties of Neurospora crassa T7H. T7H can bind the substrates only in the presence of cosubstrate, and binding of different substrates does not induce notable conformational changes. T7H exhibits comparable binding affinity for T and 5hmU, but 3-fold lower affinity for 5fU. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Our data reveal the molecular basis for substrate specificity and catalytic mechanism of T7H and provide new insights into the molecular mechanism of substrate recognition and catalysis of TET proteins.

Topics: Biocatalysis; Catalytic Domain; Fungal Proteins; Ketoglutaric Acids; Mixed Function Oxygenases; Mutagenesis; Neurospora crassa; Pentoxyl; Protein Binding; Substrate Specificity; Thymine; Uracil

The genotoxic effects of high-energy ionizing radiation have been largely attributed to the ionization of H2O leading to hydroxyl radicals and the ionization of DNA leading mostly to damage through base radical cations. However, the contribution of low-energy electrons (LEEs; ≤ 10 eV), which involves subionization events, has been considered to be less important than that of hydroxyl radicals and base radical cations. Here, we compare the ability of LEEs and high-energy X-ray photons to induce DNA damage using dried thin films of TpTpT trinucleotide as a simple and representative model for DNA damage. The main radiation-induced damage of TpTpT as measured by high-performance liquid chromatography (HPLC) with UV detection and HPLC coupled to tandem mass spectrometry analyses included thymine release (-Thy), strand breaks (pT, Tp, pTpT, TpTp, and TpT), and the formation of base modifications [5,6-dihydrothymine (5,6-dhT), 5-hydroxymethyluracil (5-hmU), and 5-formyluracil (5-fU)]. The global profile of products was very similar for both types of radiation indicating converging pathways of formation. The percent damage of thymine release, fragmentation, and base modification was 20, 19, and 61 for high-energy X-rays, respectively, compared to 35, 13, and 51 for LEEs (10 eV). Base release was significantly lower for X-rays. In both cases, phosphodiester bond cleavage gave mononucleotides (pT and Tp) and dinucleotides (pTpT and TpTp) containing a terminal phosphate as the major fragments. For base modifications, the ratio of reductive (5,6-dhT) to oxidative products (5-hmU plus 5-fU) was 0.9 for high-energy X-rays compared to 1.7 for LEEs. These results indicate that LEEs give a similar profile of products compared to ionizing radiation.

Topics: Chromatography, High Pressure Liquid; DNA Damage; Electrons; Oligonucleotides; Pentoxyl; Photons; Radiation, Ionizing; Tandem Mass Spectrometry; Thymine; Uracil

Bacteria and eukaryotes possess redundant activities that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases excise damaged bases to initiate the base excision repair pathway. hOgg1 and hNTH1, homologues of E. coli MutM and Nth, respectively, had been identified and characterized in human cells. Recent works revealed that human cells have three orthologues of E. coli Nei, hNEIL1, hNEIL2 and hNEIL3. In the present experiments, hNEIL1 protected the E. coli nth nei mutant from lethal effect of hydrogen peroxide and high frequency of spontaneous mutations under aerobic conditions. Furthermore, hNEIL1 efficiently cleaved double stranded oligonucleotides containing 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU) in vitro via beta- and delta-elimination reactions. Similar activities were detected with hNTH1. These results indicate that hNEIL1 and hNTH1 are DNA glycosylases that excise 5-foU and 5-hmU as efficiently as Tg in human cells.

Topics: Cloning, Molecular; Deoxyribonuclease (Pyrimidine Dimer); DNA Glycosylases; DNA Repair; DNA, Complementary; Escherichia coli; Humans; Hydrogen Peroxide; Mutation; Oligonucleotides; Pentoxyl; Thymine; Uracil

HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 gamma rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the gamma-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by gamma radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and gamma rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.

Topics: Calibration; Chromatography, High Pressure Liquid; Cobalt Radioisotopes; DNA; DNA Damage; DNA Repair; Dose-Response Relationship, Radiation; Gamma Rays; Models, Chemical; Nitrogen; Oxygen; Pentoxyl; Photons; Spectrophotometry; Synchrotrons; Thymine; Time Factors; Uracil; X-Rays

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

Topics: Amino Acid Sequence; Catalysis; DNA Damage; DNA Glycosylases; DNA Mutational Analysis; DNA Repair; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Pentoxyl; Structural Homology, Protein; Uracil; Uracil-DNA Glycosidase; Xenopus Proteins

To obtain the information for the role of replication protein A (RPA) on the detection of oxidized lesion in the single-stranded DNA, the binding preference of RPA purified from Xenopus egg lysate against the oligonucleotide containing one of three kinds of oxidized thymine residues, 5-formyluracil, 5-hydroxymethyluracil and 5-(1,2-dihydroxyethyl)uracil, was studied by the gel shift assay. Results of competition assay indicate that RPA preferentially binds to the oligonucleotide containing these oxidized thymine residues than the undamaged DNA.

Topics: Animals; Base Sequence; Binding Sites; DNA Damage; DNA-Binding Proteins; DNA, Single-Stranded; Oligonucleotides; Oxidation-Reduction; Pentoxyl; Pyrimidines; Replication Protein A; Thymine; Uracil; Xenopus laevis

To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU).. The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites. The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined.. The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis. The DNA polymerases incorporated only dAMP opposite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides.. These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A. It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration.

Topics: Animals; Base Pairing; DNA Glycosylases; DNA Replication; DNA Restriction Enzymes; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases; Oligonucleotides; Pentoxyl; Uracil

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

Topics: Adenine; Base Pair Mismatch; DNA Damage; DNA Glycosylases; DNA Repair; Escherichia coli; Guanine; Kinetics; Magnetic Resonance Spectroscopy; Methylnitronitrosoguanidine; Molecular Structure; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Pentoxyl; Substrate Specificity; Thymine; Uracil

Recently, an artifactual formation of a number of modified DNA bases has been alleged during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC-MS). These modified bases were 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra), and 5-formyluracil, which represent only a small percentage of more than 20 modified DNA bases that can be analyzed by GC-MS. However, relevant papers reporting the levels of these modified bases in DNA of various sources have not been cited, and differences in experimental procedures have not been discussed. We investigated the levels of modified bases in calf thymus DNA by GC-MS using derivatization at three different temperatures. The results obtained with GC/isotope-dilution MS showed that the levels of 5-OH-Cyt, 8-OH-Ade, 5-OH-Ura, and 5-OHMeUra were not affected by increasing the derivatization temperature from 23 degrees C to 120 degrees C. The level of 8-OH-Gua was found to be higher at 120 degrees C. However, this level was much lower than those reported previously. Formamidopyrimidines were readily analyzed in contrast to some recent claims. The addition of trifluoroacetic acid (TFA) adversely affected the levels of pyrimidine-derived lesions, suggesting that TFA is not suitable for simultaneous measurement of both pyrimidine- and purine-derived lesions. The data obtained were also compared with those previously published. Our data and this comparison indicate that no artifactual formation of 5-OH-Cyt, 8-OH-Ade, and 5-OHMeUra occurred under our experimental conditions in contrast to recent claims, and no prepurification of DNA hydrolysates by a tedious procedure is necessary for accurate quantification of these compounds. The artifactual formation of 8-OH-Gua can be eliminated by derivatization at room temperature for at least 2 h, without the use of TFA. The results in this article and their comparison with published data indicate that different results may be obtained in different laboratories using different experimental conditions. The data obtained in various laboratories should be compared by discussing all relevant published data and scientific facts, including differences between experimental conditions used in different laboratories.

Topics: Adenine; Animals; Artifacts; Cattle; Cytosine; DNA; Gas Chromatography-Mass Spectrometry; Guanine; Hydrolysis; Nucleotides; Oxidation-Reduction; Pentoxyl; Trifluoroacetic Acid; Uracil

Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase high-performance liquid chromatography to determine the content of the oxidation product 5-formyluracil (5-foU) in DNA. With this method, it is shown that 5-foU residues are formed with high frequency from thymine by quinone-sensitized UV-A photooxidation. Since 5-foU is also induced by ionizing radiation, it appears to be formed under conditions where thymidine radical cations are generated and react with molecular oxygen. It was previously shown that 5-foU is formed directly from [methyl-3H]thymine residues in radioactively labeled DNA by two consecutive transmutations of 3H to 3He. The theoretical basis for the kinetics of such conversion is presented in this paper, and the calculated yields are confirmed experimentally by measuring the content of 5-foU in [methyl-3H]thymine-labeled DNA aged for different time periods. Such DNA contains virtually only 5-(hydroxymethyl)uracil and 5-foU, apart from normal bases, and is therefore very useful for the investigation of repair enzyme activities involved in the repair of 5-foU-containing DNA. Using this substrate, a DNA glycosylase activity was identified in human cell extracts for the removal of 5-foU.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Base Composition; Cell Extracts; Chromatography, High Pressure Liquid; DNA; DNA Glycosylases; DNA Repair; Humans; Kinetics; Leukocytes, Mononuclear; Molecular Structure; Mutagenesis; N-Glycosyl Hydrolases; Oxidation-Reduction; Pentoxyl; Photolysis; Thymine; Ultraviolet Rays; Uracil; Vitamin K

To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizing radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-dT) was radiolabelled by nick translation with [2-14C]-thymidine triphosphate. The DNA was irradiated with X-rays and incubated with cell extract from mouse liver. Radiolabelled products released from the irradiated DNA into an ethanol-soluble fraction were analysed by reversed-phase hplc. Released 5-FU was detected as a free base during reaction with the cell extract. 5-Formyl-2'-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97 degrees C for 15 min completely abolished its ability to release 5-FU. Similar enzymatic activity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.

Topics: Animals; DNA; DNA Repair; Female; Liver; Mice; Mice, Inbred ICR; Pentoxyl; Rats; Tissue Extracts; Uracil

The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.

Topics: Adenosine Triphosphate; Ascorbic Acid; Electrophoresis, Disc; Mitosporic Fungi; Mixed Function Oxygenases; Molecular Weight; Pentoxyl; Rhodotorula; Uracil