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PubMed2006

Singlet excited-state behavior of uracil and thymine in aqueous solution: a combined experimental and computational study of 11 uracil derivatives.

The excited-state properties of uracil, thymine, and nine other derivatives of uracil have been studied by steady-state and time-resolved spectroscopy. The excited-state lifetimes were measured using femtosecond fluorescence upconversion in the UV. The absorption and emission spectra of five representative compounds have been computed at the TD-DFT level, using the PBE0 exchange-correlation functional for ground- and excited-state geometry optimization and the Polarizable Continuum Model (PCM) to simulate the aqueous solution. The calculated spectra are in good agreement with the experimental ones. Experiments show that the excited-state lifetimes of all the compounds examined are dominated by an ultrafast (<100 fs) component. Only 5-substituted compounds show more complex behavior than uracil, exhibiting longer excited-state lifetimes and biexponential fluorescence decays. The S(0)/S(1) conical intersection, located at CASSCF (8/8) level, is indeed characterized by pyramidalization and out of plane motion of the substituents on the C5 atom. A thorough analysis of the excited-state Potential Energy Surfaces, performed at the PCM/TD-DFT(PBE0) level in aqueous solution, shows that the energy barrier separating the local S(1) minimum from the conical intersection increases going from uracil through thymine to 5-fluorouracil, in agreement with the ordering of the experimental excited-state lifetime.

Models, Molecular↗

PubMed1977

NMR spectroscopy of the ring nitrogen protons of uracil and substituted uracils; relevance to A psi base pairing in the solution structure of transfer RNA.

The chemical shifts of the well-resolved ring nitrogen protons of uracil and a series of substituted uracils, including pseudouridine (5-ribosyl-uracil) and uridine (1-ribosyl-uracil) were determined using nuclear magnetic resonance (NMR). The observed chemical shifts suggest the existence of an atypical syn conformation for pseudouridine in the Apsi base pair in regulatory tRNAs in solution.

Hydrogen Bonding↗

PubMed1989

Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase.

The uracil-DNA glycosylase inhibitor gene (ugi) of the Bacillus subtilis bacteriophage PBS2 has been subcloned to a 720-base pair DNA fragment contained in pZW2-0.7 and its nucleotide sequence determined. Using nucleotide deletion analysis, we have located the cloned ugi gene along with potential regulatory elements. A promoter-like region (-10 and -35 consensus sequences) similar to other B. subtilis genes and the Shine-Dalgarno sequence characteristic of Gram-positive bacteria were both identified upstream from the initiator AUG codon. A 17-nucleotide exact inverted repeat followed by runs of adenine and thymine residues was positioned almost immediately downstream of the ochre codon. The ugi gene product was identified on sodium dodecyl sulfate-polyacrylamide gels using Escherichia coli minicells containing pZW2-0.7 and by recovering uracil-DNA glycosylase inhibitor activity following electrophoresis. The ugi gene codes for an acidic polypeptide of 9,477 molecular weight (84 amino acids) whose electrophoretic mobility was greater than predicted for a protein of this size. The mode of inhibition did not appear to involve a catalytic process nor did it directly involve inhibitor-DNA interaction. Rather, the inhibitor protein was shown to bind physically to the E. coli uracil-DNA glycosylase, forming a 36,000 molecular weight complex. This complex seems to be reversible, since inhibitor activity was recovered after heat treatment of the complex. In addition, we demonstrated that the inhibitor protein is active against uracil-DNA glycosylases isolated from several diverse biological sources but inactive against E. coli deoxyuridine triphosphatase, DNA polymerase I, and DNA polymerase alpha, beta, and gamma.

Amino Acid Sequence↗

PubMed1981

General pharmacological properties of UFT, a new type of anti-cancer agent consisting of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT) and uracil. I: Pharmacological analysis of the combined effect of FT and uracil.

The pharmacological properties of a new type of anti-cancer agent, 1-(2-tetrahydrofuryl)-5-fluorouracil (FT) and uracil (U), in a molar ratio of 1:4, were investigated and compared with those of FT, 5-fluorouracil (5-FU) and uracil. UFT at doses larger than 874.8 (FT: 270.0) mg/kg p.o. produced slight central nervous depression in mice and rats, as demonstrated by prolongation of the thiopental anesthesia, analgesic activity in the acetic acid stretching test, an anticonvulsant effect on maximal electroshock convulsions and decrease in body temperature. In rabbits UFT at a dose of 291.6 (FT: 90.0) mg/kg p.o. produced an increase in the drowsy EEG pattern. UFT at high dose produced clonic convulsions in mice and rats and cardiac fibrillation in rabbits. UFT had slight cardiorespiratory effects in anesthetized rabbits and dogs and at a dose of 291.6 (FT: 90.0) mg/kg i.v. it increased the voltage of the T-wave in ECG in anesthetized dogs. At high doses UFT inhibited gastrointestinal transit in mice and it caused emesis in dogs. At doses larger than 291.6 (FT: 90.0) mg/kg p.o. it had a diuretic effect and at higher doses it slightly increased the blood glucose level in rats. FT at equal doses to UFT had the same effects as UFT and the effect of 5-FU was similar to that of UFT. The pharmacological activity of uracil was much less potent. It was concluded that the pharmacological effect of UFT was almost entirely due to FT, however, UFT had much less effect than FT in inducing clonic convulsions, increasing the voltage of the T-waves in ECG and inducing emesis, its depressant effect is probably due to its constituent uracil.

Analgesics↗

PubMed1990

Synthesis of 1-methyl-5-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)uracil and 1-methyl-5-(3-azido-2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)uracil. The C-nucleoside isostere of 3'-azido-3'-deoxythymidine and its 2'-"up"-fluoro analogue.

1-Methyl-5-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)uracil (C-AZT), a C-nucleoside isostere of the potent anti-AIDS nucleoside 3'-azido-3'-deoxythymidine (AZT), was synthesized. 1-Methyl-2'-deoxy-5'-O-tritylpseudouridine (2a) was oxidized with CrO3/pyridine/Ac2O complex to 1-methyl-5-(5-O-trityl-beta-D-glycero-pentofuranos-3-ulosyl) uracil (12a), which was selectively reduced to 1-methyl-5-(5-O-trityl-beta-D-threo-pentofuranosyl)uracil (13a). Mesylation of 13a to 14a followed by nucleophilic displacement of the mesyloxy group with azide afforded 3'-azido-2',3'-dideoxy-5'-O-trityl-1-methylpseudoridine (15a), which was detritylated to C-AZT. In a similar manner, 1-methyl-5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (C-FMAU, a potent antiherpetic nucleoside) was converted into the 3'-azido analogue (3'-azido-C-FMAU). Both C-AZT and 3'-azido-C-FMAU, however, did not exhibit any significant inhibitory activity against HIV in H9 cells.

Antiviral Agents↗

PubMed1995

Herpes simplex virus type 1 (HSV-1) uracil-DNA glycosylase: functional expression in Escherichia coli, biochemical characterization, and selective inhibition by 6-(p-n-octylanilino)uracil.

The Herpes simplex virus type 1 (HSV-1) uracil-DNA glycosylase (UDG) is encoded by the UL2 gene. The translation from the first putative start codon of UL2 predicts a polypeptide of 334 residues, while the translation from the second start codon predicts a polypeptide of 244 residues. We have cloned and expressed the two forms of UDG, by means of the prokaryotic expression vector pMAL-c2, and both of them were enzymatically active. Furthermore, the enzymatic properties of the recombinant UDGs and of the enzyme purified from HSV-1-infected cells were similar. The two UDG polypeptides have molecular weights of 27 and 37 kDa, respectively. The 37-kDa form of recombinant UDG is consistent with the reported molecular mass of 37 kDa for the enzyme purified from HSV-1-infected cells. Both recombinant UDGs were as sensitive as UDG purified from HSV-1-infected cells to 6-(p-n-octylanilino)uracil, the most potent of a series of uracil analogs that inhibit the viral enzyme.

Base Sequence↗

PubMed1995

Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

Base Sequence↗

PubMed2000

Kinetic isotope effect studies of the reaction catalyzed by uracil DNA glycosylase: evidence for an oxocarbenium ion-uracil anion intermediate.

The DNA repair enzyme uracil DNA glycosylase catalyzes the first step in the uracil base excision repair pathway, the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA. Here we report kinetic isotope effect (KIE) measurements that have allowed the determination of the transition-state structure for this important reaction. The small primary (13)C KIE (=1.010 +/- 0.009) and the large secondary alpha-deuterium KIE (=1.201 +/- 0.021) indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) there is significant sp(2) character at the anomeric carbon. Large secondary beta-deuterium KIEs were observed when [2'R-(2)H] = 1.102 +/- 0.011 and [2'S-(2)H] = 1.106 +/- 0.010. The nearly equal and large magnitudes of the two stereospecific beta-deuterium KIEs indicate strong hyperconjugation between the elongated glycosidic bond and both of the C2'-H2' bonds. Geometric interpretation of these beta-deuterium KIEs indicates that the furanose ring adopts a mild 3'-exo sugar pucker in the transition state, as would be expected for maximal stabilization of an oxocarbenium ion. Taken together, these results strongly indicate that the reaction proceeds through a dissociative transition state, with complete dissociation of the uracil anion followed by addition of water. To our knowledge, this is the first transition-state structure determined for enzymatic cleavage of the glycosidic linkage in a pyrimidine deoxyribonucleotide.

Anions↗

PubMed2005

B cells from hyper-IgM patients carrying UNG mutations lack ability to remove uracil from ssDNA and have elevated genomic uracil.

The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.

B-Lymphocytes↗

PubMedDate not supplied

[Recognition of uracil in DNA by uracil-DNA-glycosylase from human placenta].

The affinity of different ligands (d(pA)n, d(pT)n, d(pA)nxd(pT)n, dU-containing oligonucleotides) to the uracil-DNA-glycosylase (UDG) from human placenta have been investigated. All used oligodeoxynucleotides were shown to be competitive inhibitors of uracil-DNA glycosylase toward to [3H]-uracil-DNA substrate. Minimal ligand capable to bind to the template site of the enzyme was shown to be nucleoside monophosphate (Ki(dTMP) = 30 mM, Ki(dAMP) = 10 mM). Ligand affinity increases by the factor f 1.28 and 1.36 (respectively for d(pT)n and d(pA)n) per added monomer unit according to the progression Ki[d(pN)n] = Ki(dNMP).(f)-g, where g- number of mononucleotide bases of the d(pA)n, d(pT)n. Linear dependences of -lgKi vs n have inflection point at n = 10. At n > 10 ligand affinity remain constant. Affinity of the complexes d(pA)n.d(pT)n were observed to have the analogues dependencies, but Ki was 3 fold lower than for d(pA)n with corresponding length. The Ki of duplexes containing noncomplementary residues have been determined. Insertion of dU-residues or other noncomplementary base into one of the chains of duplexes d(pA)n.d(pT)n leads to increasing of the affinity about 10-20 fold: d(pT)4(pU)(pT)5 x d(pA)10 (Ki = 6.0 MKM), d(pT)10 x d(pA)4(pU)(pA)5 (Ki = 3.0 MKM), d(pA)7(pU)(pA)7 x d(pT)7(pC)(pT)7(4.0 MKM), d(pA)7(pU)(pA)7 x d(pT)7(pG)(pT)7 (6.0 MKM), d(pA)7(pU)(pA)7 x d(pT)15 (7.0 MKM). On the basis of the data obtained the conclusion that UDG interacts with 10 mononucleotide units of DNA was reached. The contribution of 9 nonmodified base pairs of DNA into recognition of substrate containing modified base by the enzyme is about 3-4 orders of magnitude higher than the contribution of the modified base.

DNA↗

PubMed1980

High-pressure liquid chromatographic determination of ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] and GLC-mass spectrometric determination of 5-fluorouracil and uracil in biological materials after oral administration of uracil plus ftorafur.

Oral administration of uracil plus ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] has a greater antitumor effect than that of ftorafur alone. A high-pressure liquid chromatographic methods was developed for the determination of ftorafur in plasma and visceral tissues with a sensitivity of 0.025 microgram/ml or g of wet weight. A combination of GLC--mass fragmentography and GLC--mass spectrometry with total-ion monitoring was developed for the specific, simultaneous determination of 5-fluorouracil, and active metabolite, and uracil as their trimethylsilylated derivatives. The detection limits for 5-fluorouracil and uracil in the first method were 0.001 microgram/ml for plasma and 0.001--0.005 microgram/ml for visceral tissues, and those in the second method were 0.2 microgram/ml or g of wet weight. The precision and sensitivity of the assay appear to be satisfactory for determination of the levels of these compounds in plasma and visceral tissues.

Administration, Oral↗

PubMed1995

Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex.

The Bacillus subtilis bacteriophages PBS-1 and PBS-2 protect their uracil-containing DNA by expressing an inhibitor protein (UGI) which inactivates the host uracil-DNA glycosylase (UDGase) base-excision repair enzyme. Also, PBS1/2 UGI efficiently inactivates UDGases from other biological sources, including the enzyme from herpes simplex virus type-1 (HSV-1). The crystal structure of the HSV-1 UDGase-PBS1 UGI complex at 2.7 angstrum reveals an alpha-beta-alpha sandwich structure for UGI which interacts with conserved regions of UDGase involved in DNA binding, and directly mimics protein-DNA interactions observed in the UDGase-oligonucleotide complex. The inhibitor completely blocks access to the active site of UDGase, but makes no direct contact with the uracil-binding pocket itself.

Amino Acid Sequence↗

PubMed1998

A novel uracil analog, 6-chloro-5-(2-propenyl)uracil, preferentially enhances growth inhibition and differentiation of myeloid leukemia cells induced by 1alpha,25-dihydroxyvitamin D3.

The novel uracil analog, 6-chloro-5-(2-propenyl)uracil (TI90), inhibited the growth of myeloid leukemia cells and induced morphologic and functional differentiation of the cells. Although TI90 was a weak inducer of differentiation, it greatly enhanced the growth inhibition and differentiation of the leukemia cells previously induced by 1alpha,25-dihydroxyvitamin D3 (VD3) or all-trans retinoic acid (ATRA). TI90 cooperated with VD3 much more effectively than with ATRA in inhibiting cell growth and inducing differentiation. It also decreased the effective concentration of VD3 to the 10(-10) M level. On the other hand, there was no significant synergy between VD3 and the other uracil analogs. TI90 did not affect VD3 metabolism or the number and affinity of VD3 receptors (VDR) in HL-60 cells. Signals from VD3 are predominantly mediated by VDR and the ligand-activated binding of VDR to vitamin D-responsive element (VDRE) as a heterodimer with the retinoid X receptor (RXR). According to the results of a gel shift assay, TI90 enhanced the intensity of the retarded band with synthetic VDRE oligomer in the presence of VD3, suggesting that TI90 increases the number of phosphorylated receptors by inhibiting phosphatase activity, and also stimulates the formation of a functional complex of VDR with RXR.

Antimetabolites, Antineoplastic↗

PubMed1995

Sequence specificity for removal of uracil from U.A pairs and U.G mismatches by uracil-DNA glycosylase from Escherichia coli, and correlation with mutational hotspots.

The rate of removal of uracil from different positions in double-stranded DNA by uracil-DNA glycosylase from Escherichia coli varied more than 15-fold. Consensus sequences for good and poor removal were 5'-(A/T)UA(A/T)-3' and 5'-(G/C)U(T/G/C)-3', respectively. In general, the sequence context surrounding U was more important for the rate of removal than whether U was present in U.A pairs or U.G mispairs. Rates of removal of U from sites of amber mutations in the lacI gene, where mutation frequencies and deamination rates were known, indicated that the observed variation in removal is biologically significant.

Base Composition↗

PubMed1995

Cloning and expression of the uracil-DNA glycosylase inhibitor (UGI) from bacteriophage PBS-1 and crystallization of a uracil-DNA glycosylase-UGI complex.

The uracil-DNA glycosylase inhibitory protein (UGI) from the bacteriophage PBS-1 has been cloned and overexpressed. The nucleotide sequence is identical to that for the previously described PBS-2 inhibitor. The recombinant PBS-1 UGI inhibits the uracil-DNA glycosylase from herpes simplex virus type-1 (HSV-1 UDGase), and a complex between the HSV-1 UDGase and PBS-1 UGI has been crystallized. The crystals have unit cell dimensions a = 143.21 A, c = 40.78 A and are in a polar hexagonal space group. There is a single complex in the asymmetric unit with a solvent content of 62% by volume and the crystals diffract to 2.5A on a synchrotron radiation source.

Bacillus Phages↗

PubMed2006

Vibrational spectra and structure of 5,6-diamino uracil and 5,6-dihydro-5-methyl uracil by density functional theory calculations.

The molecular vibrations of 5,6-diamino uracil and 5,6-dihydro-5-methyl uracil were investigated in polycrystalline sample, at room temperature, by FT-IR and FT-Raman spectroscopies. The spectra were interpreted with the aid of normal coordinate analysis following a full structure optimization and force field calculations based on the density functional theory (DFT) using standard B3LYP/6-31G* and B3LYP/6-311+G** methods and basis set combinations. The DFT force field transformed to natural internal coordinates was corrected by a well-established set of scale factors that were found to be transferable to the title compounds. The infrared and Raman spectra were also predicted from the calculated intensities. Comparison of the simulated spectra with the experimental spectra provides important information about the ability of the computational method to describe the vibrational modes.

Crystallography↗

PubMed2002

Mutational analysis of the uracil DNA glycosylase inhibitor protein and its interaction with Escherichia coli uracil DNA glycosylase.

Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4 kDa consists of a five-stranded antiparallel beta sheet flanked on either side by single alpha helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the beta1 edge) establish key interactions with Escherichia coli UDG. The point mutations in helix alpha1 (amino acid residues 3-14) do not affect the stability of the UDG-Ugi complexes in urea. And, while the complex of the deltaN13 mutant with UDG is stable in only approximately 4M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the beta strands, conferred no such phenotype. The L23R and S21P mutations (beta1 edge) at the UDG-Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG-Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells.

Amino Acid Sequence↗

PubMed1997

Palladium-catalyzed synthesis of [E]-6-(2-acylvinyl)uracils and [E]-6-(2-acylvinyl)-1-[(2-hydroxyethoxy)methyl]uracils--their antiviral and cytotoxic activities.

[E]-6-(2-Acylvinyl)uracils and their corresponding 1-(2-hydroxyethoxy)methyl derivatives were synthesized through palladium-catalyzed reactions which involved an interesting rearrangement. Some of the acylvinyl uracils (3, 4, and 5) and the acyclonucleosides (8 and 10) showed pronounced activity against human T-lymphocyte Molt 4/C8 and CEM cells. However, they were less toxic to murine L1210 and FM3A cells. The compounds did not have any marked antiviral activity.

Animals↗