Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

5-fluorouracil (5-FU) is a widely used anticancer drug that disrupts pyrimidine nucleotide pool balances and leads to uracil incorporation in DNA, which is then recognized and removed by the uracil base excision repair (BER) pathway. Using complementary biochemical and genetic approaches we have examined the role of uracil BER in the cell killing mechanism of 5-FU. A yeast strain lacking the enzyme uracil DNA glycosylase (Ung1), which excises uracil from the DNA backbone leaving an abasic site, showed significant protection against the toxic effects of 5-FU, a G₁/S cell cycle arrest phenotype, and accumulated massive amounts of U/A base pairs in its genome (~4% of T/A pairs were now U/A). A strain lacking the major abasic site endonuclease of Saccharomyces cerevisiae (Apn1) showed significantly increased sensitivity to 5-FU with G₂/M arrest. Thus, efficient processing of abasic sites by this enzyme is protective against the toxic effects of 5-FU. However, contrary to expectations, the Apn1 deficient strain did not accumulate intact abasic sites, indicating that another repair pathway attempts to process these sites in the absence Apn1, but that this process has catastrophic effects on genome integrity. These findings suggest that new strategies for chemical intervention targeting BER could enhance the effectiveness of this widely used anticancer drug.     

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.     

Effects of 5-azacytosine in DNA on enzymic uracil excision

PBS-2 phage DNA, which contains uracil in place of thymine, was used as substrate for both purified B. subtilis uracil-DNA glycosylase and a crude extract from M. luteus. Addition of [3H]5-azacytidine to the medium after phage infection resulted in substitution of 1.2% azacytosine for cytosine in DNA. Substrate DNA was also labeled with [14C]uracil. Neither enzyme preparation released tritiated bases from DNA. Analysis by S1 nuclease digestion show no increase in single-strandedness of the modified DNA. Enzymic release of uracil by the M. luteus extract was reduced by about 50% from the substituted substrate. By contrast, the rate of uracil excision by the purified enzyme was unaffected by the presence of DNA 5-azacytosine.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.     

Facilitated transport of uracil and 5-fluorouracil,and permeation of orotic acid into cultured mammalian cells

The mode of permeation of uracil, 5–fluorouracil, and orotic acid into cells has been investigated in four established cell lines (Novikoff rat hepatoma, P388 mouse leukemia, mouse L, and Chinese hamster ovary cells) in attempts to assess the rate-determining step(s) in their incorporation into the nucleotide pool and nucleic acids. Uracil and 5–fluorouracil shared a saturable transport system (K_m = 5 to 15 mM) capable of rapid equilibration of these substrates across the cell membrane (t_1/2 at 25 in first-order range of concentration = 25 to 58 sec). Thus it seems unlikely that transport is limiting the incorporation hypoxanthine. Only the non-ionized form of fluorouracil was a substrate for the transporter; exclusion of charged pyrimidines may explain why orotate was not a substrate at physiological pH. Orotate permeated the cell membrane much more slowly (t_1/2 = 2890 to 6930 sec); its permeation was apparently non-mediated and rate-determining in the conversion of extracellular orotate to intracellular nucleotides.     

Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.     

Concerning the solvent effect in the tautomerism of uracil, 5-fluorouracil, and thymine by density-functional theory and ab initio calculations

The tautomerism of uracil, 5-fluorouracil, and thymine has been investigated in the gas phase and in solution. Electron correlation effects were included in ab initio computations at the MP2 level, and DFT calculations were performed using the B3LYP level. Full geometry optimizations were conducted at the HF/6-31G**, HF/6-31+G**, and B3LYP/6-31+G** levels. Single-point MP2/6-31+G** calculations were performed on the HF/6-31+G** optimized geometries. The influence of the solvent was examined from self-consistent reaction field calculations performed with )=2.21 (1,4-dioxane) and )=78.54 (water). The calculated relative free energies ((G) indicate that substitution of uracil at the position group does not change the relative free energy order of the uracil tautomers in the gas phase and in 1,4-dioxane (except at the MP2 level) whereas this ordering changes in water. Attachment of a fluorine atom changes the relative free energy order of uracil tautomers in the gas phase and in solution.     

An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents

1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-alpha-D-xylofuranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) uracil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically active against rotavirus infection and the growth of tumor cells.     

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.     

Role of DNA flexibility in sequence-dependent activity of uracil DNA glycosylase

Uracil DNA glycosylase (UDG) is a base excision repair enzyme that specifically recognizes and removes uracil from double- or single-stranded DNA. The efficiency of the enzyme depends on the DNA sequence surrounding the uracil. Crystal structures of UDG in complex with DNA reveal that the DNA is severely bent and distorted in the region of the uracil. This suggests that the sequence-dependent efficiency of the enzyme may be related to the energetic cost of DNA distortion in the process of specific damage recognition. To test this hypothesis, molecular dynamics simulations were performed on two sequences representing extreme cases of UDG efficiency, AUA/TAT (high efficiency) and GUG/CAC (low efficiency). Analysis of the simulations shows that the effective bending force constants are lower for the AUA/TAT sequence, indicating that this sequence is more flexible than the GUG/CAC sequence. Fluorescence lifetimes of the adenine analogue 2-aminopurine (2AP), replacing adenine opposite the uracil, are shorter in the context of the AUA/TAT sequence, indicating more dynamic base-base interaction and greater local flexibility than in the GUG/CAC sequence. Furthermore, the K(M) of Escherichia coli UDG for the AUA/TAT sequence is 10-fold smaller than that for the GUG/CAC sequence, while the k(cat) is only 2-fold smaller. This indicates that differences in UDG efficiency largely arise from differences in binding and not catalysis. These results link directly flexibility near the damaged DNA site with the efficiency of DNA repair.     

Comparative effects of ftorafur and 5-fluorouracil on DNA synthesis in rat small intestine.

The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the $\text{in}$$\text{vivo}$ incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied. 5-fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection. This degree of inhibition was sustained for at least 12 hours. Deoxyuridine incorporation was inhibited by 30 to 65% during the initial six hours after the injection of ftorafur. By 12 hours the rate of incorporation had returned to 66% of the control value. Neither drug inhibited thymidine incorporation into DNA. A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine. The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed.     

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed.     

Conformational analysis of 5-substituted uracil

The high-resolution IR-spectra of 5-nitrouracil and 5-bromouracil isolated in Ar matrices at 11 K were obtained for the first time. The conformational structure of uracil 5-substituents--thymine, 5-bromouracil, 5-nitrouracil--is calculated by the molecular mechanics and quantum--chemical MINDO/3 methods. The possibility of thymine transition to nonplanar conformations is observed.     

UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil–DNA glycosylase UNG is the major route for FU–DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil–DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU–DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.     

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 ¹H 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 T₁₂ and T₁₅ nucleotides stack upon 3′ and 5′ ends of the stem, respectively. Further, T₁₄ stacks upon both T₁₂ and T₁₅. Though U₁₃ partially stacks upon T₁₄, no stacking interaction is observed between U₁₃ and T₁₂. All the individual nucleotide bases belonging to the stem and T₁₂ and T₁₅ of the loop adopt ‘anti’ conformation with respect to their sugar moiety, while the U₁₃ and T₁₄ of the loop are in ‘syn’ conformation. The turning phosphate in the loop is located between T₁₃ and T₁₄. 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 of T₁₂ and α, β, γ, and ζ of U₁₃ and γ of T₁₄, which partially influence the local conformation of U₁₃ in U2-hairpin are all locked in ‘trans’ conformation. Such stretched out backbone conformation in the vicinity of U₁₃ 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.     

Simple liquid chromatographic method for the determination of uracil and dihydrouracil plasma levels: a potential pretreatment predictor of 5-fluorouracil toxicity

5-Fluorouracil (5-FU) is a commonly used anti-cancer drug with notable activity in clinical practice, yet it causes significant unpredictable and often serious toxicity. Both 5-FU and uracil (U) are catabolised by dihydropyrimidine dehydrogenase (DPD) to form dihydrofluorouracil (FUH(2)) and dihydrouracil (UH(2)), respectively. A means of predicting toxicity before treatment would be more valuable. Variations in dihydropyrimidine dehydrogenase (DPD) activity between patients are at least partly responsible for variable toxicity. Measurement of the UH(2) to U ratio may be a measure of pyrimidine catabolism and thus be utilised to predict subsequent toxicity. We have developed an efficient extraction and detection method using HPLC for the simultaneous measurement of UH(2) and U in plasma. A single C(18) Spherisorb ODS2 (25 cm) column using isocratic elution was utilised. U, UH(2) and the internal standard 4-chlorouracil were detected at wavelengths of 257, 220, and 268 nm, respectively. The chromatographic run time was 45 min which is half that of other methods. The detection limit was 0.02 microM for U and 0.1 microM for UH(2) using only 0.5 ml of plasma for both compounds. The basal plasma concentrations of U and UH(2) in 23 individuals ranged from 0.025 to 0.27 microM and 0.4-1.7 microM, respectively. This simple method may permit the assessment of pyrimidine catabolism, and therefore allow prediction of the toxicities associated with the use of fluorinated pyrimidines.     

The effect of 5-fluorouracil on DNA chain elongation in intact bone marrow cells

The effect of 5-fluorouracil (FUra) on DNA elongation was assessed in intact bone marrow cells that had been pulsed for 1 hr with [3H]-dThd in the absence or presence of FUra, chased in fresh media from 0 to 3 hr, and then analyzed on alkaline sucrose gradients. While DNA from control cells elongated at an average rate of 86 nucleotides per sec over a 3 hr interval, DNA from FUra-treated cells did not elongate and in contrast decreased in size over the same interval. In a parallel study to examine what happens to the FUra that was incorporated into DNA, bone marrow cells were pulsed for 1 hr with 50 microM [3H]-FUra, and then chased in fresh media from 0 to 2 hr. An aliquot of cells from each time point was lysed on an alkaline sucrose gradient to assess the size of [3H]-FUra-containing DNA, while another aliquot of cells from each time point was analyzed for radioactivity remaining in total DNA. The percentage of replicon-size DNA (greater than or equal to 100S) containing radiolabel decreased over the 2 hr chase while the percentage of small molecular weight DNA (greater than or equal to 7.2S) increased over the same interval. These changes in DNA size were accompanied by a decrease in radioactivity in total DNA. These studies suggest that excision of FUra from nascent DNA chains may prevent further elongation of DNA.