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At least 307 records · Page 17Linked to original sources
PubMed1982

Perturbations of enzymic uracil excision due to purine damage in DNA.

Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillus subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (greater than 305 nm) in the presence of isopropanol and a free radical photoinitiator introduced covalently bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by dimethylsulfate yielded 7-methylguanine. Apurinic sites were produced by gentle heating of methylated DNA. Rates of enzymic release of uracil from DNA varied among these three substrates. The Vmax was markedly decreased for DNA containing 8-(2-hydroxy-2-propyl)purines and apurinic sites but was unaffected by the presence of larger quantities of 7-methylguanine. This suggests that certain types of damaged DNA moieties may decrease the capacity for uracil excision. Therefore, interference with enzymic excision of this potentially mutagenic base may constitute a common mechanism of action of the reaction products of several unrelated DNA damaging agents.

Alkylating Agents↗
PubMed2001

Turning On uracil-DNA glycosylase using a pyrene nucleotide switch.

Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the N-glycosidic bond of 2'-deoxyuridine (2'-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2'-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.

Catalysis↗
PubMed1986

Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.

Cell Nucleus↗
PubMed1982

Properties of purified uracil-DNA glycosylase from calf thymus. An in vitro study using synthetic DNA-like substrates.

The bovine uracil-DNA glycosylase previously isolated from thymocyte nuclei was further purified by 1 order of magnitude with the aid of affinity chromatography. The final preparation was totally devoid of DNase and apurinic or apyrimidinic (AP) endonuclease activities, and it corresponded to purifications of 457-fold over the nuclear extract and of about 2000-fold over the crude tissue homogenate. Most of the general enzyme properties already described were confirmed. Furthermore, this mammalian uracil-DNA glycosylase was shown to bind specifically with polymerized and not with monomeric nucleotide compounds, while having a preference for double-stranded forms. It cleaved N-glycosyl linkages only at the deoxyuridyl units located in internal positions of polynucleotide chains. The enzyme also used RNA-DNA hybrids as functional substrates and was practically ineffective on deoxyuridyl residues at the 3'-ends of nucleic acids. The activity of the glycosylase was greatly impaired in assays with DNA substrates that contained amounts of AP sites exceeding 5 microM. The inhibitory concentrations of AP residues were about 100 times lower than those found equally effective for the other reaction product, i.e. free uracil, and were almost comparable to the Km values for deoxyuridyl nucleotides in the DNA substrates. This all appears as a modulation of the glycosylase catalysis by the relative amounts of its substrate and product structures in DNA. The data lead us to surmise that the removal of uracil from cellular DNA is functionally coupled to the expected elimination of the formed AP sites by specific endonucleases. Base-exchange and base-insertion experiments with the purified enzyme yielded negative results under various conditions. The glycosylase behaved essentially as a hydrolase which has no associated base-insertase properties and irreversibly excises uracil from DNA by a mechanism for channeling the process to the next steps of the repair pathway.

Animals↗
PubMed1985

Uridine phosphorylase from Novikoff rat hepatoma cells: purification, kinetic properties, and its role in uracil anabolism.

Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330-fold from Novikoff rat hepatoma cells by ammonium sulfate precipitation, DEAE-Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 microM, KmRib-1-P = 88 microM, KmUrd = 16 micron, and KmPi = 130 microM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 microM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose-1-phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N-(phosphonacetyl)-L-aspartate ("PALA"), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-1-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.

Animals↗
PubMed1980

Urea-acetylene dicarboxylic acid reaction: a likely pathway for prebiotic uracil formation.

A number of routes have been suggested for the prebiotic synthesis of uracil involving the reaction of urea with malic acid, propiolic acid, cyanoacetylene and others. Cyanoacetylene has been detected in the interstellar medium as well as simulated prebiotic experiments. It is therefore plausible that dicyanoacetylene and its hydrolytic product acetylene dicarboxylic acid (ADCA) may have played a role in chemical evolution. This aspect has been examined in the present work for the synthesis of uracil from ADCA and urea reaction. It was found that when ADCA reacted with urea, uracil was formed only in the presence of phosphoric acid and phosphates. ammonium phosphates gave higher yields of uracil than other phosphates. In the absence of phosphoric acid or phosphates no uracil formation took place. This type of synthesis could have taken place in prebiotic oceans which contained ammonium phosphates and other salts.

Acetylene↗
PubMed1984

Stable isotope dilution analysis of orotic acid and uracil in amniotic fluid.

Rapid, sensitive and accurate stable isotope dilution assays were developed for the measurement of orotic acid and uracil in amniotic fluid. The method utilizes [15N2]orotic acid and [15N2]uracil as internal standards, isolation by liquid partition chromatography and quantitation by chemical ionization selected ion monitoring gas chromatography-mass spectrometry. Orotic acid at a concentration of 0.26 +/- 0.05 mumol/l and uracil at a concentration of 0.55 +/- 0.13 mumol/l were detectable in normal amniotic fluid. As affected fetuses with argininosuccinate synthetase or ornithine carbamoyl transferase deficiency showed no significant elevation of orotic acid and/or uracil in their surrounding amniotic fluids, this method unfortunately seemed not to be useful for prenatal diagnosis of these inherited disorders. Nevertheless, it provides significant advantage over available methods for the quantitation of orotic acid and uracil in which the analysis of these compounds must be very accurate, highly specific and sensitive (e.g. detection of heterozygosity for ornithine carbamoyl transferase deficiency).

Amniotic Fluid↗
PubMed1986

Uracil-DNA glycosylase activity in chronic lymphoproliferative disorders.

The activity of uracil-DNA glycosylase, a repair enzyme for the excision of uracil from DNA, was studied in patients with chronic lymphoproliferative disorders and with malignant plasma cell dyscrasias. The biochemical assay was performed on mononuclear cells, isolated by density gradient centrifugation from peripheral blood, from bone marrow or from both. The activity of the uracil-DNA glycosylase of peripheral blood cells in 8/8 cases of myeloma and in 3/3 cases of Waldenström's macroglobulinemia was in the same range as in 22 non-hematological control patients, i.e. 2.4-25.1 U/mg of protein. Higher activities were found in 9/12 cases of chronic lymphocytic leukemia (CLL), in 2/4 cases of hairy cell leukemia (HCL), in 2/2 cases of chronic T-cell lymphocytosis and in the only case of small cell lymphocytic lymphoma. Follow-up of some CLL and HCL patients revealed that uracil-DNA glycosylase activity was fairly stable during the course of the disease. We conclude that malignant cells in chronic lymphoproliferative disorders are characterized by a normal or even increased capability to repair DNA, as exemplified by uracil-DNA glycosylase in this study.

Adult↗
PubMed1991

Lack of synergism in rat urinary tract carcinogenesis between prior uracil treatment and N-butyl-N-(4-hydroxybutyl)-nitrosamine administration.

Combination effects of sequential treatment with uracil prior to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) with regard to carcinogenesis in the urinary bladder and renal pelvis were investigated in male F344 rats. Uracil was administered for 15, 10 or 5 weeks followed by BBN for 23 weeks, the total observation time being 40 weeks. Carcinoma(s) and papilloma(s) were induced in the urinary tract by the 15-week uracil treatment independent of subsequent BBN administration. It was concluded that uracil administration prior to BBN treatment is not associated with any synergistic effects, although both uracil and BBN alone exerted carcinogenicity.

Animals↗
PubMed1994

Initiation-stage enhancement by uracil of N-ethyl-N-hydroxyethylnitrosamine-induction of kidney carcinogenesis in rats.

The present study was conducted to clarify the effects of N-ethyl-N-hydroxyethylnitrosamine (EHEN) and uracil in combination on renal carcinogenesis in female F344 rats. Group 1 animals (n = 30) received a 3-week simultaneous administration of 0.05% EHEN and 3% uracil, and group 2 (n = 26) 0.05% EHEN with a lower 1.5% dose of uracil. Group 3 (n = 30) was treated with 0.05% EHEN alone and group 4 (n = 28) received only 3% uracil for 3 weeks. In all the above four groups, the rats were given basal diet and water without chemical addition for a 48-week period after the 3-week treatment period. Group 5 (n = 33) received no chemicals for the entire 52 weeks. At the end of week 52, renal adenocarcinomas were found in 53%, 31% and 13% of the rats in groups 1, 2, and 3, respectively. The present study thus demonstrated that simultaneous administration of uracil enhances the occurrence of EHEN-induced renal adenocarcinomas in rats.

Adenocarcinoma↗
PubMed1990

Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.

Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.

DNA Glycosylases↗
PubMed1992

Uracil-induced urolithiasis in the urinary bladder of rats is irritation-dependent.

The correlation between urolithiasis due to uracil administration and epithelial proliferation in the urinary bladder was investigated in male F344 rats. Animals, 6 weeks old at the start, which ingested 3% uracil alone in the diet for 4 weeks developed urolithiasis and papillomatosis of the urinary bladder, whereas addition of NaCl, particularly at high concentration (10%), to the 3% uracil in the diet blocked production of both. In contrast, NaHCO3 was without influence on either calculi or proliferative changes. These results indicate that uracil-induced epithelial proliferation is due to direct mechanical irritation by calculi, and not to any chemical stimulus presented by uracil itself.

Animals↗
PubMed1985

Identification of Escherichia coli by detection of hydroquinone and uracil in the urine system.

For rapid identification of Escherichia coli, changes of urinary metabolites incubated with E. coli were investigated by gas chromatography--mass spectrometry. Hydroquinone and uracil were detected and the normal urinary constituent 4-deoxythreonic acid was found to diminish in urine incubated with E. coli. Hydroquinone could not be detected in urine incubated with Klebsiella pneumoniae, Serratia marcescens or Pseudomonas aeruginosa. Although uracil was detected in normal urine, urine incubated with E. coli showed an increased uracil level. Urine incubated with K. pneumoniae, S. marcescens or P. aeruginosa evidenced no such change. A decrease of 4-deoxythreonic acid was noted in urine incubated with S. marcescens or P. aeruginosa. In 7.0 X 10(7) cells of E. coli, 0.33-2.36 micrograms of hydroquinone and 13.4-42.0 micrograms of uracil were detected after 3 h of incubation at 38 degrees C, and production was not changed after 4, 5 or 8 h of incubation. These results suggest that the detection of hydroquinone and uracil in urine is useful for rapid identification of E. coli.

Escherichia coli↗
PubMed2004

The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase.

The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA. We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached. We find that the bipartite mimics of TS2 are superior to the TS1 mimics. These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.

Amides↗
PubMed2005

Surface-enhanced Raman scattering and density functional theory calculation of uracil on gold and silver nanoparticle surfaces.

The adsorption structure of uracil on gold and silver nanoparticle surfaces has been comparatively studied by means of surface-enhanced Raman scattering (SERS). Uracil appeared to assume a perpendicular orientation with respect to the surfaces. The presence of the nu(CH) band in the SERS spectra indicated a vertical orientation of the aromatic ring of uracil on Au and Ag. The density functional theory (DFT) calculation was performed at the levels of B3LYP and MP2 to estimate the energetic stability of the N3- and N1-deprotonated tautomers and their vibrational frequencies on the surfaces. Almost all the vibrational bands in the SERS spectra at high concentrations could be ascribed to the N3-deprotonated uracil. The N3-deprotonated tautomer was predicted to be more favorable on Au than on Ag from the DFT calculation. The metal-N bond distance was assumed to be shorter for Au than for Ag upon adsorption of uracil.

Gold↗
PubMed1978

Binding sites between platinum (II) and uracil derivatives.

The complexes of triammineplatinum with uracil, 6-methyluracil, or uridine were prepared in aqueous solution at pH 7. The reaction of uracil with triammineplatinum gave two complexes at the same time. One was a complex in which triammineplatinum displaced a proton from uracil and coordinated to the N(3) position, and the other the complex coordinated to the N(1) position by displacing a proton. When triammineplatinum was treated with 6-methyluracil or uridine in aqueous solution at pH7, only a complex coordinated to the N(3) position was obtained. The ultraviolet (UV), NMR, and infrared (IR) spectral data provide useful information for determining the binding site of these complexes. The UV and IR spectral behaviors of the complex coordinated to the N(3) of uracil are very similar to those of 3-methyluracil. The NMR spectrum of the complex coordinated to the N(1) of uracil exhibits satellite peaks of 195Pt-proton and its coupling constant (39 Hz) gives good evidence for determining the binding site at the N(1) position.

Chemical Phenomena↗
PubMed2001

Properties and functions of human uracil-DNA glycosylase from the UNG gene.

The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences. PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2. Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease. The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out. Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine. These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E. coli chromosome and were used to examine biological consequences of AP sites. It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination. However, one major role of UNG2 is to remove misincorporated dUMP residues. Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell. We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution.

Animals↗
PubMed1997

Potential signalling roles for UTP and UDP: sources, regulation and release of uracil nucleotides.

Increasing evidence for receptors for uracil nucleotides has focused interest on specific signalling mechanisms involving UTP and UDP. At least three metabotropic P2 receptors are stimulated by uracil nucleotides with equal or greater potency than by adenine nucleotides, and there might be ionotropic receptors as well. Regulation of uridine and uracil nucleotide levels is important when considering the receptor-mediated effects of these compounds. Cells can synthesize uracil nucleotides de novo or by salvage of uridine. UTP made from salvage might be preferentially used for RNA synthesis in the nucleus, while UTP synthesized de novo seems to be used for UDP-sugar and CDP-phospholipid production. UTP from both pathways can enter a free UTP pool, from which UTP can be released from cells. UTP and UDP can stimulate pyrimidinoceptors, but metabolism by ecto-nucleotidases limits their effects. Alternatively, UTP might be a substrate for ecto-protein kinases, and this could contribute to its extracellular regulation. Cells can reclaim uridine, using nucleoside transport processes, following dephosphorylation of UTP, UDP and UMP. In this article Christopher Anderson and Fiona Parkinson discuss how understanding the processes that regulate uridine and uracil nucleotide concentrations will enhance our ability to manipulate UTP/UDP signalling pathways for pharmacological effect.

Animals↗