Identification and partial characterization of rabbit brain deoxyuridine 5′-triphosphatase

Adult rabbit brain contains the enzymatic machinery to convert deoxyuridine to deoxyuridine triphosphate (dUTP). Although dUTP as dUMP can be readily incorporated into DNA in place of thymidine monophosphate, we detected no (3H)dUMP in newly synthesized (3H)DNA in adult rabbit brain after the intraventricular injection of (3H)deoxyuridine. Only (3H)thymidine was detected. The probable explanation for the lack of incorporation of uracil into adult rabbit brain DNA is the presence of a specific, high affinity dUTPase which converts dUTP to dUMP and PP. After homogenization and ammonium sulfate fractionation of adult rabbit brain (35 to 75% saturation), a high affinity, specific dUTPase was detected in the dialyzed enzyme preparation. The Km and Vmax of the dUTPase were 0.2 microM and 36 pmol/mg protein/min, respectively. No high affinity dUTPase activity was detectable in liver. In brain, another enzyme hydrolyzed dUTP and dTTP (NTPase( to their respective diphosphates. NTPase, unlike dUTPase, was not sensitive to heating at 65 degrees C for five minutes. Thus, brain, like other tissues, contains a high affinity, specific dUTPase presumably to "sanitize" the cells of dUTP and, thus, protect the integrity of newly synthesized DNA.     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target.

dUTP pyrophosphatase catalyses hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and inorganic pyrophosphate (PPi). Elimination of dUTP is vital since its misincorporation into DNA by DNA polymerases can initiate a damaging iterative repair and misincorporation cycle, resulting in DNA fragmentation and cell death. The anti-tumour activity of folate agonists and thymidylate synthase inhibitors is thought to rely on dUTP misincorporation. Furthermore, retroviral cDNA production may be particularly susceptible to the effects of dUTP misincorporation by virtue of the error-prone nature of reverse trans criptase. Consequently, dUTPase activity is an ideal point of intervention in both chemotherapy and anti-retroviral therapy. In particular, the dUTPase encoded by a human endogenous retrovirus (HERV-K) has been suggested to complement HIV infection and so is an attractive target for specific inhibition. Hence, we used site photoaffinity labelling, site-directed mutagenesis and molecular modelling to assign catalytic roles to the conserved amino acid residues in the active site of the HERV-K dUTPase and to identify structural differences with other dUTPase enzymes. We found that dUTP photoaffinity labelling was specific for a beta-hairpin motif in HERV-K dUTPase. Mutagenesis of aspartate residues Asp84 and 86 to asparagine within this beta-hairpin showed the carboxylate moiety of both residues was required for catalysis but not for dUTP binding. An increase in the pKa of both aspartate residues brought about by substitution of a serine residue with a glutamate residue adjacent to the aspartate residues increased activity by a factor of 1.67 at pH 8.0, implicating general base catalysis as the enzyme's catalytic mechanism. Conservative mutagenesis of Tyr87 to Phe resulted in a sevenfold reduction of dUTPase activity and a 3.3-fold reduction in binding activity, whilst substitution with an isoleucine residue totally abolished both catalytic activity and dUTP binding, suggesting that binding/activity is dependent on an aromatic side-chain at the base of the hairpin. Comparison of a homology-based three-dimensional model structure of HERV-K dUTPase with a crystallographic structure of the human dUTPase revealed displacement of a conserved alpha-helix in the HERV-K enzyme causing expansion of the HERV-K active site. This expansion may be responsible for the ability of the HERV-K enzyme to hydrolyse dTTP and bind the bulkier dNTPs in contrast to the majority of dUTPases which are highly specific for dUTP. Knowledge of the dUTPase catalytic mechanism and the distinctive topography of the HERV-K active site provides a molecular basis for the design of HERV-K dUTPase-specific inhibitors.     

Depletion of dimeric all-alpha dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-alpha NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.     

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66 microM while the k(cat) was 12.3 s(- 1). dUDP was also efficiently hydrolysed although the specificity constant, k(cat)/K(m), was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue alpha,beta imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3'- and 5'-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.     

African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg(2+), the preferred cation, and has an apparent K(m) for dUTP of 1 microM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vDeltaE165R) has been obtained. Growth kinetics showed that vDeltaE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.     

Partial purification of deoxyuridine triphosphate nucleotidohydrolase and its effect on DNA synthesis in isolated HeLa cell nuclei

Deoxyuridine triphosphate nucleotidohydrolase (EC 3.6.U.23) has been partially purified from HeLa S3 cells, and found to have an apparent molecular weight of 50--55 000 by gel filtration under non-denaturing conditions. The enzyme is specific for the hydrolysis of dUTP, requires Mg2+ and is inhibited by EDTA. The apparent Km for dUTP is 0.1 microM. Isolated HeLa cell nuclei were treated with dUTPase before pulse-labelling with [3H]dTTP which also had been pretreated with dUTPase. This pretreatment changed neither the total amount nor the size of the primary DNA pieces. A role for dUTP incorporation in their genesis can therefore be excluded and these primary DNA pieces are considered to be true intermediates in discontinuous DNA replication.     

Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.     

Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type 1. Purification and characterization of induced enzyme

A deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is induced in KB cells infected with herpes simplex virus type 1 (HSV-1) was purified approximately 175-fold using affinity, hydrophobic, adsorption, and ion-exchange chromatography techniques. Of the nucleoside triphosphates commonly found in DNA and RNA, only dUTP acted as a substrate for the enzyme, and the apparent Km was 4 microM. While the HSV-1-induced dUTPase exhibited activity in the absence of added divalent cations, the activity was stimulated by Mg2+ and inhibited by EDTA. The inhibition caused by EDTA was reversed by Mg2+, Co2+, or Mn2+. The HSV-1-induced dUTPase was also inhibited by hydroxymercuribenzoate and to a lesser degree by pyrophosphate but not by orthophosphate. The molecular weight of the enzyme was estimated to be 53,000, and its isoelectric point was 5.8. The enzyme exhibited maximal activity over the pH range of 6.5-8.5. The enzyme was thermolabile at 45 degrees C, exhibiting a t1/2 of 35 min. The HSV-1-induced dUTPase was distinguishable from the KB dUTPase by its elution pattern on the various chromatography matrixes, isoelectric point, migration in polyacrylamide gels, thermostability, and response to divalent cations.     

Backbone nuclear magnetic resonance assignment of human deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase)

Nuclear-associated deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is an enzyme that hydrolyses deoxyuridine 5′-triphosphate (dUTP) to the monophosphate, thereby controlling the dUTP levels of the organism, which is essential for survival. Further, dUTPase is up-regulated in many cancers. Thus, dUTPase is a highly interesting potential drug target. We report, for the first time, the near complete nuclear magnetic resonance (NMR) spectroscopy ¹⁵N/¹³C/¹H backbone assignment of the 3 × 164 amino acids homo-trimer human dUTPase. Previously, only a handful backbone resonances belonging to the flexible C-terminus has been published for any protein in the dUTPase family.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

人细胞核dUTPase的克隆表达及其酶学活性

以阿尔茨海默病 (Alzheimer’sdisease ,AD)患者脑cDNA文库质粒为模板 ,用PCR方法扩增得到人细胞核dUTP焦磷酸酶 (dUTPase)的cDNA ,将其克隆到谷胱甘肽 S 转移酶 (GST)融合表达载体pGEX 4T 1中 ,并在大肠杆菌BL2 1中获得高效表达 .表达的融合蛋白GST dUTPase经过谷胱甘肽 Sepharose 4B亲和层析 ,凝血酶酶切和SephacrylS 10 0纯化 ,得到高纯度dUTPase蛋白 .通过SDS PAGE ,氨基酸组成分析 ,N端氨基酸序列测定以及HPLC测Mr 结果与期望值一致 .通过检测该酶水解dUTP释放的焦磷酸 (PPi)来测定表达产物dUTPase蛋白及GST dUTPase融合蛋白的酶活性 ,发现两蛋白都具有正常的酶水解dUTP活性 ,但融合蛋白的活性比dUTPase蛋白低 7～ 8倍 .同时研究了Mg2 +和EDTA对酶活性的影响     

DNA repair synthesis-related enzymes during spermatogenesis in the mouse

To assess whether uracil DNA glycosylase and dUTP nucleotidohydrolase (dUTPase) can be involved in repair-type DNA synthesis associated to crossing-over or induced by UV and X-ray treatments, we have studied these enzyme activities in male mouse germ cells at specific stages of differentiation.Although the highest uracil DNA glycosylase activity was observed in dividing germ cells (spermatogonia and preleptotene spermatocytes), some activity was also detected in meiotic (3.5%) and post-meiotic (1.0%) cells with a relative maximum of activity at pachytene stage (4.7%) when meiotic crossing-over takes place. These findings suggest that uracil DNA glycosylase is involved, in this biological system, in DNA replication and in repair-type DNA synthesis.dUTPase is present at all the stages of spermatogenesis studied but, unlike thymidylate synthetase which is mainly associated with replicating germ cells, dUTPase activity is maximal in spermatocytes at pachytene stages. The data reported suggest that, in this biological system, the main role of dUTPase is to degrade dUTP to prevent misincorporation of uracil into DNA during crossing-over, rather than to participate in the biosynthetic pathway of dTTP.     

金属硫蛋白-3对dUTPase调节dUTP细胞毒性作用的影响

金属硫蛋白-3（MT-3）,又称神经生长抑制因子,是一种脑特异性金属硫蛋白。人细胞核dUTP焦磷酸酶（dUTP pyrophosphatase, dUTPase）是最近在脑中研究发现的能与人金属硫蛋白-3（human metallothionein-3,hMT-3）相互作用的一个蛋白,两者共同作用具有神经元生长抑制活性。为了探讨hMT-3对dUTPase调节dUTP引起的细胞毒性作用的影响,通过基因转染HEK293细胞观察细胞在dUTP中的生长情况,发现共转染hMT-3和dUTPase基因的细胞比单转染dUTPase或hMT-3基因的细胞具有更强的对dUTP细胞毒性的耐受能力;同时在大肠杆菌BL21中表达重组蛋白,测定hMT-3在dUTPase水解dUTP中的作用,结果显示重组蛋白hMT-3可以促进dUTPase对dUTP的水解。结果均初步证实了hMT-3对dUTPase抵抗dUTP引起的正常细胞死亡有一定的协同作用,为进一步研究dUTPase及其相互作用蛋白hMT-3在化疗中的应用提供理论依据。     

Early Biochemical Events Occurring After Infection of Bacillus cereus 569-SP1 with Bacteriophage GSW

After infection of Bacillus cereus 569-SP1 with the 5-hydroxymethyluracil-containing phage GSW, new dTTPase, dUTPase, and dUMP-hydroxymethylase activities appear. No significant changes in activities of other pyrimidine ribonucleoside or 2'-deoxyribonucleoside triphosphate nucleotidohydrolases were detected. dUTP and dUMP inhibit the dTTPase activity, whereas dTTP failed to inhibit dUTPase activity. The K(m) value for the substrate dUTP is 10(-4) M and for dTTP is 4.85 x 10(-4) M. Thymidylate synthetase activity is inhibited only when cells are infected during the late lag or very early log phases of growth; when cells are infected with phage during mid-log, thymidylate synthetase activity is unaffected. The data support the suggestion that, although phage GSW may inhibit an otherwise expected increase in activity of thymidylate synthetase, it fails to affect the already existing activity. The data presented do not allow discrimination as to whether the phage specifies inhibition of de novo synthesis of thymidylate synthetase or the increase in activity of already existing but not fully expressed enzyme.     

New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection.     

Deoxyuridine triphosphate nucleotidohydrolase: distribution of the enzyme in various rat tissues

The distribution of deoxyuridine triphosphate nucleotidohydrolase (dUTPase) [EC 3.6.1.23] in the cytosol of various rat tissues was investigated by measuring the enzyme activity and by immunochemical analyses. Among nine rat tissues, thymus, and spleen showed the highest activities of the enzyme per gram of tissue, while intestine, stomach, lung and liver showed very low levels. Rabbit antibodies directed against purified dUTPase of anemic rat spleen showed reactivity with partially purified dUTPases from other rat tissues such as thymus, testis, or regenerating liver. Immunotitration and immunoblot experiments also indicated that the dUTPases in various rat tissues had very similar antigenicity and apparently the same subunit molecular size (Mr = 19,500), suggesting that the enzyme lacks organ-specificity. Immunoblot analysis of dUTPase protein with crude extracts from various rat tissues showed a similar distribution to that of the enzyme activity. No immuno-reactive band corresponding to the dUTPase was detected in intestine, although intestinal mucosa has been recognized as an actively proliferating tissue.     

PHOSPHATASE ACTIVITY AND CELLULAR DIFFERENTIATION IN PHLEUM ROOT MERISTEM

Using 9 different organic phosphate substrates as alternatives in a standardized 5′-nucleotidase histochemical test system, enzyme activity patterns were recorded for timothy grass root epidermis. At least 4 different phosphatases were distinguished on the bases of substrate specificity, reaction rate, tissue distribution, and response to inhibitors. Except with adenosine-3′-monophosphate, all activities were restricted to the 300-mμ-long root tip meristem. These enzyme activities were associated with the earliest phases of differentiation of the epidermal hair and hairless cell initials. The distribution of activities was not associated with the same cell type in each part of the meristem. Little activity was found with most substrates in the undifferentiated cells of the 0-100μ zone; alternating active hairless and inactive hair cell initials predominated in the 100-200μ segment; and active hair–inactive hairless sister cells formed the principal pattern in the 200-300μ segment of the meristem. The data showed that a particular enzyme activity was associated with a specific cell type only in relation to that cell's position along the differentiation gradient of the entire tissue. But, within a meristem segment, a specific cell type might act differently from its neighbors, depending on its mitotic capacity. This complex of physiological dependence and independence of a cell type on tissue ontogeny was cited as a characteristic of the phenomenon of cellular differentiation superimposed on tissue differentiation gradients.     

Crystallization and preliminary investigation of single crystals of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized. The crystals belong to the trigonal space group R3 and diffract beyond 2 A. The native dUTPase crystals and a mercury derivative are stable in the X-ray beam and are suitable for a high resolution X-ray structure analysis.