Disease relevance of ECs4515

• Here we report the three-dimensional structure of E. coli dUTPase determined by X-ray crystallography at a resolution of 1.9 A [1].
• The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5'-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid [2].
• Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins [3].
• dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency [3].
• By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro [3].

High impact information on ECs4515

• The enzyme dUTPase catalyses the hydrolysis of dUTP and maintains a low intracellular concentration of dUTP so that uracil cannot be incorporated into DNA. dUTPase from Escherichia coli is strictly specific for its dUTP substrate, the active site discriminating between nucleotides with respect to the sugar moiety as well as the pyrimidine base [1].
• The nucleotide sequence of the dUTPase structural gene, dut, of Escherichia coli has been determined [4].
• The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods [5].
• The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP [6].
• These mutant strains have now been found to be defective in deoxyuridinetriphosphate diphosphohydrolase (dUTPase; deoxyuridinetriphosphatase, EC 3.6.1.23), the enzyme that catalyzes the hydrolysis of dUTP to dUMP and PPi [7].

Chemical compound and disease context of ECs4515

• Bacteriophage T5 induces a deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) activity during infection of Escherichia coli [2].
• The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region [8].
• The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase [9].
• Selective modification of one (of three) tyrosine residue per enzyme monomer leads to inactivation of dUTPase of the retrovirus equine infectious anemia virus (EIAV) [10].
• We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP [11].

Biological context of ECs4515

• The DNA sequence predicts a polypeptide chain of 150 amino acid residues (mol. wt. 16 006) corresponding in size and composition to the purified dUTPase subunit [4].
• Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase [3].
• The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone [12].
• The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81-->Ser81 and Thr84-->Arg84, in the highly conserved motif III of the proteins [13].
• Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus [12].

Anatomical context of ECs4515

• Restoration of the biological activity of in vitro synthesized phi X DNA by transfection of ung- spheroplasts or dUTPase treatment [14].
• PURPOSE: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity [15].
• The recombinant protein was compared with the native dUTPase similarly purified from HSV-1-infected Vero cells (African green monkey kidney fibroblasts) [16].

Associations of ECs4515 with chemical compounds

• A decrease in dUTPase as a consequence of a sof or dut mutation may result in the increased incorporation of uracil into DNA [7].
• This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step [17].
• By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides [18].
• By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase [17].
• The recombinant protein has dUTPase activity and requires Mg(2+) for optimal activity, while it retains some activity in the presence of other divalent cations [13].

Other interactions of ECs4515

• Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE [19].
• The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii [20].

Analytical, diagnostic and therapeutic context of ECs4515

• Through the molecular cloning of DNA, cells were obtained that could produce a 300-fold increased level of deoxyuridine triphosphatase (dUTPase) [21].
• As a consequence of Tyr-93 derivatization, the Mg2+-dependent interaction between the substrate-analogue dUDP and E. coli dUTPase becomes impaired as shown by circular dichroism spectroscopy, here presented as a tool for monitoring ligand binding to the active site [10].
• We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets [22].

References