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The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP. With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1. Km passed through a broad minimum in the neutral pH range with values approaching 10(-7) M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase.UDP complex.

The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [gamma-32P]5-azido-dUTP and [gamma-32P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.

Glutamate 60 of thymidylate synthase coordinates a hydrogen bond network important in proton transfer reactions to and from the substrate dUMP. The E60A and E60L mutants of Lactobacillus casei thymidylate synthase catalyzed tritium exchange from [5-3H]dUMP for solvent protons faster than dTMP formation, indicating accumulation of a steady-state intermediate and a change in partitioning of the intermediate. A covalent complex consisting of E60A or E60L thymidylate synthase, dUMP, and the cofactor CH2H4 folate was isolated on SDS-polyacrylamide gel electrophoresis and shown to be chemically and kinetically competent to form dTMP. These results provide proof of the formation of a covalent steady-state intermediate in the reaction pathway of thymidylate synthase and demonstrate that the rate-determining step in the mutants occurs during conversion of the covalent intermediate to dTMP.

Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a Kd of 720 nM. The binding of the substrate to the second subunit (site B) was much weaker, and the Kd could not be determined by this method. However, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative Kd value for each subunit varied substantially. For example, formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dideazafolate bound to both sites with similar Kd values, whereas D1694Glu4 bound to subunit A with a higher affinity (Kd = 1.0 nM) than to subunit B (Kd = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (Kd approximately 0.1 nM) compared with subunit A (Kd approximately 400 nM). Enzyme inhibition kinetic analyses showed that the Ki values of 1843U89 were quite low (0.1 nM) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (i.e. both the Km for the folate substrate and the Vmax were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.

We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.

Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.

E-5-(2-Bromovinyl)-2'-deoxyuridylate (BrvdUMP), the first metabolite in the processing of the antiviral agent E-5-(2-bromovinyl)-2'-deoxyuridine (BrvdUrd), is an excellent alternate substrate for dTMP synthetase. The nucleophilic catalyst of the enzyme adds to the 6-position of the heterocycle and converts the normally inert 5-bromovinyl group of BrvdUMP to a reactive allylic bromide in which both carbons of the side chain are susceptible to nucleophilic attack. These centers react with nucleophiles in the reaction mixture, 2-mercaptoethanol and water, to give three diastereomeric products which have been isolated and characterized. Possible implications of these findings as related to the mechanism and selectivity of BrvdUrd as an antiviral agent are discussed.

Bacteriophage T5 induces a deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) activity during infection of Escherichia coli. A T5 mutant (T5 dut) unable to induce this dUTPase activity has been isolated. Although this mutant is viable, the E. coli dUTPase activity is not sufficiently active to exclude uracil from the progeny DNA and about 3% of the thymine is replaced by uracil. When the mutant is grown in an E. coli dut host about 12% of the thymine in the progeny DNA is replaced by uracil. T5 phage containing 12% uracil can replicate in uracil-DNA glycosylase-deficient (ung) hosts with high efficiency, but fail to replicate in ung+ hosts. The amount of thymine replaced by uracil in the progeny produced in dut hosts is nearly independent of the ung genotype, indicating that the host uracil-DNA glycosylase-dependent repair pathway is not operating efficiently to remove uracil from T5 progeny DNA.

Thymidylate synthetases of human and bacterial origin form a tightly bound complex with the substrate dUMP in the presence of pteroyltriglutamate. This complex and the weaker enzyme . dUMP binary complex can be isolated and conveniently assayed by nitrocellulose disc filtration using [6-3H]dUMP as the radioactive ligand. Intact thymidylate synthetase . dUMP . pteroyltriglutamate complex can be obtained by gel filtration chromatography on Sephadex G-25, but the binary enzyme . dUMP complex dissociates under the same conditions. Scatchard plots show the presence of two nonequivalent dUMP binding sites on the enzyme for the pteroyltriglutamate complex, with dissociation constants of 5 and 95 nM compared to 730 nM for the binary complex. The implications of these findings for folate analog inhibition of thymidylate synthetase are discussed.

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).

The amounts of intracellular nucleotides of dThd and dUrd were measured in cultured human lymphoblasts with and without treatment with methotrexate (plus hypoxanthine) to inhibit thymidylate synthetase. dTTP fell from approximately 40 pmol/10(6) cells (untreated) to approximately 1 pmol/10(6) cells with drug treatment. dUMP, increased approximately 10(3)-fold with methotrexate. dUTP was not detected in the cell without drug treatment (less than or equal to 0.3 fmol/10(6) cells), but was easily measured at approximately 0.2 pmol/10(6) cells in drug-inhibited cells. Thus the ratio dUTP/dTTP incrased from less than or equal to 10(-5) in untreated cells to approximately 0.2 in cells treated with methotrexate, indicating that dUMP may be incorporated in substantial amounts into the DNA of the drug-treated cells. The magnitude of these effects on nucleotide pools suggests the possibility that the normal nucleolytic mechanism for removal of dUMP from DNA may play a part in the toxic effects on the cell that result from depression of thymidylate synthetase activity.

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.

5-Nitro-2'-deoxyuridylate (NO2dUMP) is a potent mechanism-based inhibitor of dTMP synthetase. After formation of a reversible enzymeinhibitor complex, there is a rapid first order loss of enzyme activity which can be protected against by the nucleotide substrate dUMP. From studies of model chemical counterparts and the NO2dUMPdTMP synthetase complex, it has been demonstrated that a covalent bond is formed between a nucleophile of the enzyme and carbon 6 of NO2dUMP. The covalent NO2dUMPenzyme complex is sufficiently stable to permit isolation on nitrocellulose membranes, and dissociates to give unchanged NO2-dUMP with a first order rate constant of 8.9 x 10(-3) min-1. Dissociation of the complex formed with [6-3H]NO2dUMP shows a large alpha-secondary isotope effect of 19%, verifying that within the covalent complex, carbon 6 of the heterocycle is sp3-hybridized. The spectral changes which accompany formation of the NO2dUMPenzyme complex support the structural assignment and, when used to tritrate the binding sites, demonstrate that 2 mol of NO2dUMP are bound/mol of dimeric enzyme. The interaction of NO2dUMP with dTMP synthetase is quite different than that of other mechanism-based inhibitors such as 5-fluoro-2'-deoxyuridylate in that it neither requires nor is facilitated by the concomitant interaction of the folate cofactor, 5,10-CH2-H4folate, and that the covalent complex formed is unstable to protein denaturants.

In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.

In contrast to previous reports, fluorescence and ultraviolet difference spectroscopy are shown to reflect the interaction of the inhibitor, 5-fluoro-2'-deoxyuridylate, with thymidylate synthetase isolated from amethopterin-resistant Lactobacillus casei. Analysis of the quenching of protein fluorescence upon nucleotide binding yields a value of 2.1 X 10(5) M-1 for the association constant for binary complex formation. The ultraviolet difference spectrum for the nucleotide-enzyme complex exhibited a broad trough with the greatest loss of absorbance centered at 275 nm. Results of previous circular dichroic studies of nucleotide-enzyme binary complexes showed apparently parallel changes in ellipticity in the 267-269-nm and 290-nm regions, which were interpreted to reflect coordinated alteration in nucleotide and enzyme structure, respectively. When examined by difference circular dichroic spectroscopy, the nucleotide-enzyme interaction is accompanied by a substantial loss in ellipticity from 250 to 300 nm which is greatest at 280 nm. We interpret the results obtained from the three light spectroscopic techniques as indications of subtle alterations in the environments of certain tyrosine and tryptophan residues in the enzyme which are caused by the association of 5-fluoro-2'-deoxyuridylate with the enzyme.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.

In view of removing lesions in DNA produced by the deamination of cytosine to uracil, uracil-DNA glycosylases were anticipated to be ubiquitous. However, an analogous activity in Drosophila melanogaster was not detected. Instead, a nuclease was identified that acts specifically upon DNA containing uracil. The cleavage of uracil-containing DNA by the nuclease generates acid-soluble oligonucleotides in a reaction which can be inhibited by pretreatment of the DNA with Escherichia coli uracil-DNA glycosylase. Uracil-containing DNA with either A:U base pairs or G:U base pairs were susceptible to cleavage by the nuclease, whereas other damaged DNA substrates were not. The nuclease activity is transient and appears only in third instar larvae, with other developmental stages of Drosophila lacking significant levels of the nuclease.