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PubMed1997

Photochemical production of uracil quantified in bromodeoxyuridine-substituted SV40 DNA by uracil DNA glycosylase and a lysyl-tyrosyl-lysine tripeptide.

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechst dye 33258 results in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB) yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis. Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is approximately 5-fold more efficient than SSB formation. Formation of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.

Bromodeoxyuridine↗
PubMed2000

Structural basis for uracil DNA glycosylase interaction with uracil: NMR study.

Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.

DNA↗
PubMed1982

Uracil-DNA glycosylase. Purification and properties of uracil-DNA glycosylase from Micrococcus luteus.

A uracil-DNA-glycosylase from Micrococcus luteus has been purified more than 3,000-fold. The enzyme preparation appears homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is devoid of nonspecific endonucleases, specific endonucleases for apurinic and apyrimidinic sites, 3-methyladenine or 7-methylguanine-DNA-glycosylases. It behaves as a monomer protein of 19,400 daltons. It has an isoelectric point of 7.0 +/- 0.1. It has an optimal activity between pH 5.0 and 7.0. It has no cofactor requirement and is not inhibited by EDTA. Uracil-DNA-glycosylase is highly specific for DNA containing dUMP residues, releasing uracil as product of the reaction. It is 2-fold more active on single-stranded DNA than on double-stranded DNA. If it releases uracil dimers from ultraviolet-irradiated PBS1 DNA, it is at the threshold of the detection. The apparent Km is 7 X 10(-8) M, and uracil acts as a noncompetitive inhibitor with a Ki of 3.2 X 10(-4) M. Cis-syn cyclogbutadiuracil also is a potent inhibitor, while some analogs, produced by x-irradiation of uracil and thymine, are weak inhibitors. Spermine, between 10 and 400 microM, increases the enzymatic activity by 50% and is not inhibitory at other concentrations. Spermidine activates the enzyme at concentrations of 40 to 120 microM, but becomes inhibitory at 200 and 400 microM. A new finding is that drugs which intercalate in DNA, such as ethidium bromide and ellipticine, cause a 2- to 2.5-fold activation of this enzyme activity. The concentrations giving maximal activation depend on the drug. The enzyme does not behave as a processive enzyme during uracil excision.

DNA↗
PubMed2006

Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress.

Oxidative stress in the brain may cause neuro-degeneration, possibly due to DNA damage. Oxidative base lesions in DNA are mainly repaired by base excision repair (BER). The DNA glycosylases Nei-like DNA glycosylase 1 (NEIL1), Nei-like DNA glycosylase 2 (NEIL2), mitochondrial uracil-DNA glycosylase 1 (UNG1), nuclear uracil-DNA glycosylase 2 (UNG2) and endonuclease III-like 1 protein (NTH1) collectively remove most oxidized pyrimidines, while 8-oxoguanine-DNA glycosylase 1 (OGG1) removes oxidized purines. Although uracil is the main substrate of uracil-DNA glycosylases UNG1 and UNG2, these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence of multi-protein BER complexes in nuclei and mitochondria. Extracts from nuclei and mitochondria were both proficient in complete uracil-BER in vitro. BER assays with immunoprecipitates demonstrated that UNG2-EYFP, but not UNG1-EYFP, formed complexes that carried out complete BER. Although apurinic/apyrimidinic site endonuclease 1 (APE1) is highly enriched in nuclei relative to mitochondria, it was apparently the major AP-endonuclease required for BER in both organelles. APE2 is enriched in mitochondria, but its possible role in BER remains uncertain. These results demonstrate that nuclear and mitochondrial BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines.

Bacterial Proteins↗
PubMed2002

The acidity of uracil and uracil analogs in the gas phase: four surprisingly acidic sites and biological implications.

The gas phase acidities of a series of uracil derivatives (1-methyluracil, 3-methyluracil, 6-methyluracil, 5,6-dimethyluracil, and 1,3-dimethyluracil) have been bracketed to provide an understanding of the intrinsic reactivity of uracil. The experiments indicate that in the gas phase, uracil has four sites more acidic than water. Among the uracil analogs, the N1-H sites have deltaH(acid) values of 331-333 kcal mol(-1); the acidity of the N3 sites fall between 347-352 kcal mol(-1). The vinylic C6 in 1-methyluracil and 3-methyluracil brackets to 363 kcal mol(-1), and 369 kcal mol(-1) in 1,3-dimethyluracil; the C5 of 1,3-dimethyluracil brackets to 384 kcal mol(-1). Calculations conducted at B3LYP/6-31+G* are in agreement with the experimental values. The bracketing of several of these sites involved utilization of an FTMS protocol to measure the less acidic site in a molecule that has more than one acidic site, establishing the generality of this method. In molecules with multiple acidic sites, only the two most acidic sites were bracketable, which is attributable to a kinetic effect. The measured acidities are in direct contrast to in solution, where the two most acidic sites of uracil (N1 and N3) are indifferentiable. The vinylic C6 site is also particularly acidic, compared to acrolein and pyridine. The biological implications of these results, particularly with respect to enzymes for which uracil is a substrate, are discussed.

Acids↗
PubMed1999

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.

Binding Sites↗
PubMed1994

Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil DNA glycosylase.

Kinetic parameters for uracil DNA glycosylase (E. coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined. Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides. This is mainly because of the favourable Vmax value of the enzyme for single-stranded substrates. More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient. The poor efficiency with which uracil is excised from loop regions is a result of both increased Km and lowered Vmax values. This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins. In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.

Base Sequence↗
PubMed2006

Effects of microsolvation on uracil and its radical anion: uracil(H2O)n (n = 1-5).

Microsolvation effects on the stabilities of uracil and its anion have been investigated by explicitly considering the structures of complexes of uracil with up to five water molecules at the B3LYPDZP++ level of theory. For all five systems, the global minimum of the neutral cluster has a different equilibrium geometry from that of the radical anion. Both the vertical detachment energy (VDE) and adiabatic electron affinity (AEA) of uracil are predicted to increase gradually with the number of hydrating molecules, qualitatively consistent with experimental results from a photodetachment-photoelectron spectroscopy study [J. Schiedt et al., Chem. Phys. 239, 511 (1998)]. The trend in the AEAs implies that while the conventional valence radical anion of uracil is only marginally bound in the gas phase, it will form a stable anion in aqueous solution. The gas-phase AEA of uracil (0.24 eV) was higher than that of thymine by 0.04 eV and this gap was not significantly affected by microsolvation. The largest AEA is that predicted for uracil(H2O)5, namely, 0.96 eV. The VDEs range from 0.76 to 1.78 eV.

Anions↗
PubMed1977

Biochemical mechanism of uracil uptake regulation in Escherichia coli B. Allosteric effects on uracil phosphoribosyltransferase under stringent conditions.

The regulation of uracil uptake in bacteria was studied in bacteriophage T4-infected cells, where host-specific, stable RNA synthesis is completely shut-off by phage, and where phage-specific RNA synthesis, which is not stringently regulated, could be followed by a continuous incorporation of uracil. This incorporation into phage RNA was found to be dependent on the allelic state of the rel gene and it was thus severely restricted under stringent conditions. This was not the case with adenine, which was incorported into RNA to almost the same extent under stringent and relaxed conditions, respectively. The inhibition of uracil uptake under proceeding RNA formation, which was furthermore found to be reversed by addition of chloramphenicol, indicated a specific mechanism governing the cellular entry of uracil. This is suggested to involve the allosteric regulation of uracil phosphoribosyltransferase (EC 2.4.2.9.). The enzyme was partially purified by ammonium sulfate precipitation and gel chromatography. The dependence on GDP and GTP as positive effectors was demonstrated. The stimulatory effect of GTP was abolished in vitro by the addition of guanosine 5'-diphosphate 3-diphosphate, which is known to accumulate during amino acid starvation in stringent bacteria. The reversible inactivation of the enzyme by dilution suggested a subunit structure of uracil phosphoribosyltransferase.

Adenine↗
PubMed2000

Escherichia coli uracil DNA glycosylase: NMR characterization of the short hydrogen bond from His187 to uracil O2.

Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 10(12)-fold over the solution reaction. The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using (1)H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 A) H bond between His187 N(epsilon)(2) and uracil O2. The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 10(5)-fold slower than free imidazole at pH 7.5 and pH 10, respectively. Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil. The excellent correspondence of the pK(a) for the disappearance of the H bond (pK(a) = 6.3 +/- 0.1) with the previously determined pK(a) = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 [Drohat, A. C., and Stivers, J. T. (2000) J. Am. Chem. Soc. 122, 1840-1841]. Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of phi = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor-acceptor pK(a) mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (phi < 1) and increased bending frequencies (phi > 1) relative to the O-H bonds of water. The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed.

Anions↗
PubMed2001

The role of leucine 191 of Escherichia coli uracil DNA glycosylase in the formation of a highly stable complex with the substrate mimic, ugi, and in uracil excision from the synthetic substrates.

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic. Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi. In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.

Amino Acid Substitution↗
PubMed1993

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Animals↗
PubMed1999

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.

Amino Acid Sequence↗
PubMed1999

Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.

Amino Acid Sequence↗
PubMed2000

Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP. G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.

Base Pair Mismatch↗
PubMed2006

A uracil-DNA glycosylase inhibitor encoded by a non-uracil containing viral DNA.

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5'-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.

Bacillus Phages↗
PubMed1993

Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds.

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry. Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex. Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation. Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4. Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively. While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid. Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism. The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM. The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1. We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex.

Binding Sites↗
PubMed1994

Effect of gastrectomy on the pharmacokinetics of tegafur, uracil, and 5-fluorouracil after oral administration of a 1:4 tegafur and uracil combination.

The effects of gastrectomy on the pharmacokinetics of UFT, a combined oral preparation of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil at a molar ratio of 1:4, were examined in 26 patients with macroscopic Stage I gastric cancer. In all, 200 mg UFT (in terms of tegafur) was given to 17 patients who underwent partial gastrectomy (9 cases of Billroth I reconstruction, 8 cases of Billroth II reconstruction) and to 9 patients who underwent total gastrectomy with modified Roux-en-Y reconstruction. Before the operation, the area under the curve (AUC) for tegafur, uracil, and 5-fluorouracil (5-FU) was 79.28 +/- 26.88, 4.41 +/- 1.78, and 0.51 +/- 0.20 micrograms h ml-1, respectively. Partial (Billroth I and II) and total gastrectomy did not alter the AUC of tegafur, and partial gastrectomy using the Billroth I and II methods decreased the AUCs of uracil and 5-FU during the first 2 weeks postoperation. However, plasma levels of uracil and 5-FU reverted to preoperative values at 3 months postsurgery. Our findings show that when UFT is prescribed for patients treated in the early postoperative period following partial gastrectomy for cancer, dose increases and the timing of administration should be given close attention.

Administration, Oral↗