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PubMed2005

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs.

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.

Base Pair Mismatch↗

PubMed1981

Uridine and uracil transport in Escherichia coli and transport-deficient mutants.

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterize the transport mechanism of uracil and uridine. Mutant U-, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR-, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U-UR- isolated from a culture of mutant U-, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR-R- was isolated from a culture of mutant UR-, and is resistant to a high concentration of showdomycin. This mutant is defective for transport of intact uridine and addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U- and UR-, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these processes.

Adenine↗

PubMed1997

Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats.

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.

Animals↗

PubMed2004

Barrier-free intermolecular proton transfer induced by excess electron attachment to the complex of alanine with uracil.

The photoelectron spectrum of the uracil-alanine anionic complex (UA)(-) has been recorded with 2.540 eV photons. This spectrum reveals a broad feature with a maximum between 1.6 and 2.1 eV. The vertical electron detachment energy is too large to be attributed to an (UA)(-) anionic complex in which an intact uracil anion is solvated by alanine, or vice versa. The neutral and anionic complexes of uracil and alanine were studied at the B3LYP and second-order Møller-Plesset level of theory with 6-31++G(*) (*) basis sets. The neutral complexes form cyclic hydrogen bonds and the three most stable neutral complexes are bound by 0.72, 0.61, and 0.57 eV. The electron hole in complexes of uracil with alanine is localized on uracil, but the formation of a complex with alanine strongly modulates the vertical ionization energy of uracil. The theoretical results indicate that the excess electron in (UA)(-) occupies a pi(*) orbital localized on uracil. The excess electron attachment to the complex can induce a barrier-free proton transfer (BFPT) from the carboxylic group of alanine to the O8 atom of uracil. As a result, the four most stable structures of the uracil-alanine anionic complex can be characterized as a neutral radical of hydrogenated uracil solvated by a deprotonated alanine. Our current results for the anionic complex of uracil with alanine are similar to our previous results for the anion of uracil with glycine, and together they indicate that the BFPT process is not very sensitive to the nature of the amino acid's hydrophobic residual group. The BFPT to the O8 atom of uracil may be relevant to the damage suffered by nucleic acid bases due to exposure to low energy electrons.

Alanine↗

PubMed1992

Survival of phage M13 with uracils on one or both DNA strands.

The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage. Uracils carry the same genetic information as the thymines. Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG). Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG. Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides. The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (-) strand only. Incorporation of 246 uracils into the (+) strand leads to approximately 30% or approximately 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively. However, when uracils are distributed over both strands a sharp decrease in survival occurs. This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.

Base Sequence↗

PubMed1996

Affinity purification and comparative analysis of two distinct human uracil-DNA glycosylases.

Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by the Bacillus subtilis bacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS-PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently post-translationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal that in vitro expressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS-PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.

Affinity Labels↗

PubMed2004

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes.

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.

Cells, Cultured↗

PubMed2002

Uracil in human DNA from subjects with normal and impaired folate status as determined by high-performance liquid chromatography-tandem mass spectrometry.

A sensitive and selective method for determination of the uracil content in human DNA was first developed on the basis of high-performance liquid chromatography-tandem mass spectrometry. Uracil was excised from DNA using uracil DNA glycosylase. The released uracil was derivatized with 4-bromomethyl-7-methoxycoumarin, thereby forming bis-N,N'-(4-methylene-7-methoxycoumaryl)-uracil (uracil-MMC). 15N2-Uracil was used as an internal standard. The analytes were separated on an Adsorbsphere XL ODS column. A SCIEX API III tandem mass spectrometer equipped with a turbo ion-spray interface was used as the detector. Multiple reaction monitoring using the parent --> product ion combinations of m/z 489 --> 232 and 491 --> 233 were used to detect uracil-MMC and the internal standard, respectively. The detection limit for this assay is <1.0 x 10(-10) mol/L uracil, and the linearity is from 1.0 x 10(-10) to 2.5 x 10(-6) mol/L. The method was used for determination of uracil in human DNA. Our data show that the uracil levels in human DNA isolated from peripheral white blood cells did not differ between subjects with folate deficiency and subjects with normal red cell folate levels.

Base Composition↗

PubMed1998

Proton stoichiometry of the overexpressed uracil symport of the yeast Saccharomyces cerevisiae.

Information about the molecular biology of the uracil carrier of Saccharomyces cerevisiae is now available but its properties as a symport are unexplored. We have now studied its proton stoichiometry at pH 6.5, 4.8 and 4.2, in a yeast strain overexpressing the symport and unable to metabolize uracil. After the depletion of cellular ATP, uracil uptake followed an approximately exponential time course to reach a plateau. Proton uptake was then indistinguishable from the basal rate shown in controls without added uracil. It was concluded that more than 87% of the uracil flux through the system was coupled to symported protons. Because the basal rate was a significant fraction of the total rate of proton uptake at pH 4.2 or 4.8, the ratio of the proton and uracil flows could not be defined uniquely during the initial near-linear phase of uptake. However, the average rate of proton uptake during increasing time intervals up to 120 s was shown to be a linear function of the corresponding average rate of uracil uptake. This relationship, defining approx. 90% of the eventual uracil uptake, was used to deduce the mean proton stoichiometry as approx. 3 at pH 4.2, falling to near 2 at pH 6.5. The rate of uracil uptake increased 4-fold when the negative proton gradient (Delta mu(H)) acting across the plasma membrane increased from 60 to 200 mV. The rate fell markedly after depolarization by KCl. The maximum uracil gradient (Delta mu(S)) was less affected by depolarization. The ratio Delta mu(S)/Delta mu(H) fell from approx. 2 to near 1 as Delta mu(H) increased in the above range. In contrast with the starved yeast, uracil accumulation during energy metabolism followed a pump-leak model.

Biological Transport↗

PubMed1978

Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome.

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [(3)H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I (polA(-)) and polA(-) strains that are also defective in uracil N-glycosidase, an enzyme required for the excision-repair of uracil in DNA (polA(-)ung(-)). Little or no difference was detected in the level of Okazaki fragments in the polA(-) strain as compared with the polA(-)ung(-) strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N-glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.

Chromosomes↗

PubMed1999

Properties of uracil transport by vegetative mycelium of Trichoderma viride.

The transport of radioactively labelled uracil into submerged mycelium of T. viride was measured by means of a membrane filtration technique. It was found to be time-dependent (up to 90 min) and concentration-dependent (up to 8 mmol l-1). Its concentration dependence was biphasic and consisted from the saturatable part (at the uracil concentration below 0.2 mmol l-1) with KM = 0.08 +/- 0.02 mmol l-1 and Vmax = 1.74 +/- 0.3 nmol (mg dry wt.)-1 h-1, and from the region at higher uracil concentration which showed only a weak saturatability with the substrate. The transport measured in the saturatable part of the curve was also pH- and temperature-dependent. The optimal pH was between 5.4 and 6.4 and the optimal temperature was at 37 degrees C. The activation energy of 54 kJ mol-1 and the temperature quotient of Q10 = 2.1 could be calculated from the temperature dependence. The entry of uracil was in part inhibited by nucleobases and their analogues, nucleosides, nucleotides and amino acids. The inhibitors had similar inhibitory efficiency about 50% at 0.2 mmol l-1. 3,3',4',5-tetrachlorosalicylanilide (TCS), the uncoupling agent, significantly inhibited the uracil transport, but its inhibitory efficiency decreased upon increasing the uracil concentration. Ionophore antibiotics valinomycin and monensin also inhibited the uracil transport. Inhibitors of RNA-polymerase, rifamycin and rifampicin were without effect. The results suggest that at low uracil concentrations (below 0.2 mmol l-1), its transport is mediated by a carrier and is driven by the electrochemical potential of protons. At higher uracil concentrations, the transport may be driven by the concentration difference of uracil with the contribution of the protonmotive force. It is feasible that inhibitors of uracil transport tested exert their inhibition by the dissipation of the driving force rather than by the direct competition with the substrate-binding site.

Biological Transport↗

PubMed1990

A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.

Base Composition↗

PubMed1996

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.

Animals↗

PubMed1988

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver.

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.

Animals↗

PubMed1998

Fidelity and mutational specificity of uracil-initiated base excision DNA repair synthesis in human glioblastoma cell extracts.

The fidelity of DNA synthesis associated with uracil-initiated base excision repair was measured in human whole cell extracts. An M13mp2 lacZalpha DNA-based reversion assay was developed to assess the error frequency of DNA repair synthesis at a site-specific uracil residue. All three possible base substitution errors were detected at the uracil target causing reversion of opal codon 14 in the Escherichia coli lacZalpha gene. Using human glioblastoma U251 whole cell extracts, approximately 50% of the heteroduplex uracil-containing DNA substrate was completely repaired, as determined by the insensitivity of form I DNA reaction products to cleavage by a combined treatment of E. coli uracil-DNA glycosylase and endonuclease IV. The majority of repair occurred by the uracil-initiated base excision repair pathway, since the addition of the bacteriophage PBS2 uracil-DNA glycosylase inhibitor protein to extracts significantly blocked this process. In addition, the formation of repaired form I DNA molecules occurred concurrently with limited DNA synthesis, which was largely restricted to the HinfI DNA fragment initially containing the uracil residue and specific to the uracil-containing DNA strand. Based on the reversion frequency of repaired M13mp2 DNA, the fidelity of DNA repair synthesis at the target was determined to be about one misincorporated nucleotide per 1900 repaired uracil residues. The major class of base substitutions propagated transversion mutations, which were distributed almost equally between T to G and T to A changes in the template. A similar mutation frequency was also observed using whole cell extracts from human colon adenocarcinoma LoVo cells, suggesting that mismatch repair did not interfere with the fidelity measurements.

Bacillus Phages↗

PubMed2000

Oral uracil and Ftorafur plus leucovorin: pharmacokinetics and toxicity in patients with metastatic cancer.

PURPOSE: To assess the pharmacokinetics of Ftorafur (tegafur, FT), 5-fluorouracil (5-FU), and uracil in 31 cancer patients who were enrolled in phase I studies of oral uracil and FT (UFT). The correlation between pharmacokinetic parameters and toxic effects of UFT was evaluated. METHODS: Uracil and FT were orally administered in a 4:1 molar ratio at FT doses of 200-400 mg/m2 per day. Patients also received leucovorin at 150 mg/day. Daily doses were divided into three doses and administered at 8-h intervals for 28 consecutive days. Plasma FT concentrations were measured by high-performance liquid chromatography, and plasma 5-FU and uracil concentrations were determined using gas chromatography-mass spectrometry. National Institutes of Health Common Toxicity Criteria were used for assessment of toxicity. RESULTS: The concentrations of FT, 5-FU, and uracil showed wide interpatient variations. Maximum plasma concentrations (Cp(max)) of all three compounds were achieved in 0.3 to 4.0 h. At the various study doses, the terminal half-life (t 1/2beta) of FT ranged from 3.9 to 5.9 h, the area under the concentration-versus-time curve (AUC0-6h) ranged from 16,220 to 52,446 (ng/ml)h, the total clearance (ClT) ranged from 100 to 175 ml/min, and the steady-state volume of distribution (Vd(ss)) ranged from 18.3 to 28.7 l. The 5-FU generated from FT had an apparent distribution half-life (t 1/2alpha) and an apparent elimination half-life (t 1/2beta) of 0.3-1.3 h and 4.9-7.0 h, respectively. The AUC0-6h of 5-FU ranged from 120 to 325 (ng/ml)h. Uracil had a t 1/2alpha of 0.2-0.5 h and the level quickly returned to the endogenous level. The AUC0-6h for uracil ranged from 605 to 3764 (ng/ml)h, the ClT ranged from 3225 to 7748 ml/min, and the Vd(ss) ranged from 341 to 1354 l. The Cp(max) and AUC0-6h of both FT and uracil were significantly correlated with FT doses (P-values of 0.0244 and 0.0112) and with uracil doses (P-values of 0.0346 and 0.0083), respectively. In addition to interpatient variations, intrapatient variations were also observed in six patients who had pharmacology studies done on days 1 and 26+/-2 at the same study dose. We found that the repeated treatment with UFT caused cumulative increases in the values of Cp(max), Ctrough, and AUC0-6h of FT and 5-FU. The major toxic effects observed were diarrhea and nausea and vomiting. The occurrence of these toxic effects correlated significantly with the Cp(max) and AUC0-6h of 5-FU. CONCLUSIONS: The pharmacology studies showed that FT and uracil were readily absorbed orally and that FT was rapidly converted to 5-FU. The preliminary findings suggest that determination of plasma levels of 5-FU after oral administration of UFT may help predict subsequent toxic effects.

Tegafur↗

PubMed1986

Inhibition of the intestinal transport of uracil by hexoses and amino acids.

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.

Amino Acids↗

PubMed1991

Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase.

DNA repair of genetic information is an essential defense mechanism, which protects cells against mutation and transformation. The biochemistry of human DNA repair is in its beginning stages. Our research has concentrated on the enzymes involved in the removal of atypical bases from DNA. We present information on the identification and characterization of a cDNA isolate encoding uracil-DNA glycosylase. Uracil-DNA glycosylase was purified to homogeneity from HeLa S3 cells and used to generate polyclonal antibodies. These antibodies were in turn used to isolate a uracil-DNA glycosylase specific cDNA from a human T cell (Jurkat) lambda-gt11 library. The identity of this 1.25 kb cDNA was verified using in vitro transcription and translation systems to generate specific uracil-DNA glycosylase activity. Sequence data revealed a 327 amino acid open reading frame, which encodes a protein with a predicted molecular weight of 35351. No significant amino acid homology was found between this human uracil-DNA glycosylase and the glycosylases of yeast, Escherichia coli, herpes simplex virus, or a recently identified 26,000 Da species of human uracil-DNA glycosylase. This apparent lack of homology prompted an investigation of uracil-DNA glycosylase in a variety of eukaryotic species. Western analysis demonstrated the presence of a 36 kDa uracil-DNA glycosylase protein in human fibroblast, human placental and Vero cell extracts. Interestingly, these antibodies did not detect glycosylase protein in Chinese hamster ovary (CHO) or mouse NIH3T3 fibroblast cells. Under conditions of reduced stringency, Southern blot analysis of BamHI digested DNA from human fibroblasts, human placental cells and Vero cells revealed common 12 kb and 3 kb fragments. In contrast, using the same reduced stringency protocol, 6 and 8 kb fragments for CHO and NIH3T3 DNA were seen, respectively, as well as a common 3 kb fragment. Under more stringent wash conditions, the common 3 kb band was absent in all samples analyzed, and no hybridization signal was detected from DNA of hamster or mouse origin. The lack of immunological reactivity between the human uracil-DNA glycosylase and the rodent forms is therefore reflected at the genetic level as well. This distinction in human and CHO hybridization patterns enabled us to localize this human uracil-DNA glycosylase cDNA to chromosome 5 by somatic cell hybridization.

Amino Acid Sequence↗