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EC Tree
The enzyme appears in viruses and cellular organisms
Synonyms
gene 4 protein, t7 helicase, dttpase, t7 dna helicase,
more
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desoxythymidine-5'-triphosphatase
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thymidine triphosphate nucleotidohydrolase
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dTTPase
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dTTP + H2O = dTDP + phosphate
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phosphorous acid anhydride hydrolysis
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dTTP nucleotidohydrolase
Also acts, more slowly, on dUTP and UTP.
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dCTP + H2O
dCDP + phosphate
dTTP + H2O
dTDP + phosphate
dTTP + H2O
dTMP + diphosphate
dTTP/dCTP + H2O
dTDP/dCDP + phosphate
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-
?
dUTP + H2O
dUDP + phosphate
dUTP + H2O
dUMP + diphosphate
UTP + H2O
UDP + phosphate
additional information
?
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wild type gp4 helicase unwinds the duplex DNA
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?
dCTP + H2O
dCDP + phosphate
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?
dCTP + H2O
dCDP + phosphate
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very poor substrate
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?
dCTP + H2O
dCDP + phosphate
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60% activity compared with dTTP as substrate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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mechanisms of DNA-dependent and-independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA, overview
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?
dTTP + H2O
dTDP + phosphate
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the lysines on loop II are essential for ssDNA binding
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?
dTTP + H2O
dTDP + phosphate
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dTTP hydrolysis is coupled to DNA unwinding, dependence of dTTP hydrolysis rates on dsDNA stability, the force-producing step during DNA unwinding is not associated with dTTP binding, but dTTP hydrolysis or phosphate release
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?
dTTP + H2O
dTDP + phosphate
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dTTP as part of DNA molecules, activity assay using four different DNA substrates with the same GC content of 5%, but varying lengths of duplex regions, fluorescent substrates, mechanochemistry and kinetics of the DNA-unwinding reaction, overview
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?
dTTP + H2O
dTDP + phosphate
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Gp4 binds, as a hexamer, to single-stranded DNA in the presence of dTTP and translocates in a 5' to 3' direction along the DNA strand using the energy derived from the hydrolysis of dTTP
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTMP + diphosphate
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?
dTTP + H2O
dTMP + diphosphate
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?
dUTP + H2O
dUDP + phosphate
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?
dUTP + H2O
dUDP + phosphate
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50% activity compared with dTTP as substrate
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?
dUTP + H2O
dUDP + phosphate
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50% activity compared with dTTP as substrate
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?
dUTP + H2O
dUMP + diphosphate
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?
dUTP + H2O
dUMP + diphosphate
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70% activity compared with dTTP as substrate
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?
UTP + H2O
UDP + phosphate
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20% activity compared with dTTP as substrate
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?
UTP + H2O
UDP + phosphate
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20% activity compared with dTTP as substrate
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?
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dTTP + H2O
dTDP + phosphate
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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mechanisms of DNA-dependent and-independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA, overview
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-
?
dTTP + H2O
dTDP + phosphate
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dTTP hydrolysis is coupled to DNA unwinding, dependence of dTTP hydrolysis rates on dsDNA stability, the force-producing step during DNA unwinding is not associated with dTTP binding, but dTTP hydrolysis or phosphate release
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-
?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
dTTP + H2O
dTDP + phosphate
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?
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Ca2+
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weak activator
Ca2+
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1 mM, 70% activation compared with Mn2+
Mg2+
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50 mM MgCl2, maximum activity
Mg2+
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1 mM, 80% activation compared with Mn2+
Mn2+
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Mn2+
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reverses inhibitory effect of EDTA
Zn2+
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effective activator
Zn2+
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1 mM, very weak activation
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dCTP
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competitive inhibitor with dTTP as substrate, Ki: 0.25 mM
diphosphate
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with dTTP or dUDP as substrate
dTDP
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with dTTP or dUTP as substrate
dUDP
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with dTTP or dUTP as substrate
dUTP
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with dTTP as substrate
para-hydroxy-mercuribenzoate
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no inhibition in presence of 25 mM beta-mercaptoethanol
additional information
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different purin- and pyrimidinnucleotide tested
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EDTA
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complete inhibition
EDTA
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with 0.2-2.0 mM EDTA 80% loss of activity
Zn2+
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causes complete inhibition in absence of EDTA
Zn2+
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complete inhibition
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single-stranded DNA
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the presence of single-stranded DNA stimulates the rate of dTTP hydrolysis about 100fold in the case of wild type enzyme
sulfhydryl compound
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required for activity of the partially purified enzyme
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Adenomatous Polyposis Coli
6-gene promoter methylation assay is potentially applicable for prostate cancer clinical staging based on urine collection following prostatic massage.
Carcinoma
Cloning and expression of soluble recombinant human esophageal cancer related gene 4 protein and its inhibitory effect on tumor growth in vitro and in vivo in esophageal carcinoma.
Colorectal Neoplasms
Human serum deoxythymidine-5'-triphosphatase activity as a parameter in the diagnosis and follow-up of large bowel carcinoma.
Esophageal Neoplasms
Cloning and expression of soluble recombinant human esophageal cancer related gene 4 protein and its inhibitory effect on tumor growth in vitro and in vivo in esophageal carcinoma.
Head and Neck Neoplasms
Tumor markers in the diagnosis and follow-up of head and neck cancer: role of CEA, CA 19-9, SCC, TK, and dTTPase.
Hepatitis C
Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability.
Infections
Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.
Infections
Synthesis of thymine and alpha-putrescinylthymine in bacteriophage phi W-14-infected Pseudomonas acidovorans.
Neoplasms
Cloning and expression of soluble recombinant human esophageal cancer related gene 4 protein and its inhibitory effect on tumor growth in vitro and in vivo in esophageal carcinoma.
Neoplasms
Elevated dTTPase activities in sera of patients with advanced cancer.
Neoplasms
Human serum deoxythymidine-5'-triphosphatase activity as a parameter in the diagnosis and follow-up of large bowel carcinoma.
Neoplasms
Serum deoxythymidine-5'-triphosphatase activity in lymphoproliferative disorders of men and mice.
Neoplasms
Tumor markers in the diagnosis and follow-up of head and neck cancer: role of CEA, CA 19-9, SCC, TK, and dTTPase.
Rotavirus Infections
Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV.
Stroke
Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7.
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0.125
dTTP/dCTP
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equimolar
additional information
additional information
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dTTP hydrolysis kinetics are measured in real time using the phosphate-binding protein that acts as a sensitive sensor for fast detection of low concentrations of phosphate, overview
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0.038
dTTP
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0.06
dTTP
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mutant enzyme R493Q, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.07
dTTP
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mutant enzyme R493A/N468A, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.07
dTTP
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wild type enzyme, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
2.6
dTTP
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wild type enzyme, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
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0.05
dTTP
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mutant enzyme N468A, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.05
dTTP
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mutant enzyme R493A/N468A, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.06
dTTP
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mutant enzyme N468A, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.08
dTTP
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mutant enzyme R493Q, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.22
dTTP
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mutant enzyme R493Q, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.22
dTTP
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wild type enzyme, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
0.35
dTTP
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mutant enzyme R493A/N468A, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
20
dTTP
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wild type enzyme, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
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3.8
dTTP
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mutant enzyme R493Q, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
5
dTTP
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mutant enzyme R493A/N468A, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
5.7
dTTP
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wild type enzyme, in the absence of DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
7.7
dTTP
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wild type enzyme, in the presence of M13 single-stranded DNA, in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 50 mM potassium glutamate, at 37°C
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0.25
dCTP
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competitive inhibitor with dTTP as substrate
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7.5
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DNA binding and dTTPase assays at
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6 - 9
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no activity at pH 6, very low activity at pH 9
7.5 - 10
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measured in Tris-buffer and glycine-buffer
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37
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37
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DNA binding and dTTPase assays at
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0
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25% of activity compared with activity at 37°C
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brenda
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brenda
infected with phage phi
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brenda
infected with phage SP82G
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brenda
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brenda
the enzyme is encoded on gene 4
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brenda
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brenda
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associated
brenda
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100000
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gel filtration in presence of 1% Triton
48500
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gel filtration and sucrose density gradient centrifugation
23000
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SDS-PAGE
23000
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2 * 23000, SDS-PAGE
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hexamer or heptamer
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in the presence of dTTP Gp4 oligomerizes to form hexamers and heptamers
additional information
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all subunits are catalytic
dimer
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2 * 23000, SDS-PAGE
dimer
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2 * 24000, SDS-Page, gel filtration
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E343D
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the mutant does not hydrolyze dTTP
E343N
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the mutant does not hydrolyze dTTP
N468A
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the mutant hydrolyzes dTTP at rates comparable with wild type enzyme. N468A hydrolyzes nearly 4fold less efficiently than the wild type enzyme in the absence of single-stranded DNA and is not stimulated by the presence of single-stranded DNA
R493A/N468A
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the mutant hydrolyzes dTTP at rates comparable with wild type enzyme and is not stimulated by the presence of single-stranded DNA
E343Q
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site-directed mutagenesis, the mutant is inactive, dTTP hydrolysis and DNA unwinding activities are abolished, but the mutant shows increased single-stranded DNA binding affinity compared to the wild-type enzyme, overview
E343Q
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the mutant does not hydrolyze dTTP
R493Q
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the mutant hydrolyzes dTTP at rates comparable with wild type enzyme and is not stimulated by the presence of single-stranded DNA
R493Q
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the mutant maintains only the basal level of dTTPase activity but does not exhibit activation in the dTTP hydrolysis activity in the presence of single-stranded DNA
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9
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loss of activity above pH 9
210043
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12 - 23
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no loss of activity within 1 h
30 - 37
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stability starts to decline above 30°C and breaks down at 37°C
50
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loss of activity in phage infected cells, no loss of activity in wild type cells
50
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no differences in inactivation between wild type and phage infected cells
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-80°C, 10 mM Tris/HCl, pH 7,8, 5 mM mercaptoethanol, stable on freezing and thawing two times
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use of preparative SDS-PAGE, for small amounts of pure protein
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partial purification
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expressed in Escherichia coli HMS-174(DE3) cells
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renaturation after preparative SDS-PAGE
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medicine
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potential prognostic marker for different diseases e.g. leukemia
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Roscoe, D.H.
Thymidine triphosphate nucleotidohydrolase: a phage-induced enzyme in Bacillus subtilis
Virology
38
520-526
1969
Bacillus subtilis
brenda
Dahlman, N.; Ueckermann, C.
Properties of four different desoxy-thymidine-5'-triphosphate hydrolyzing enzymes in human serum
Biochem. Int.
5
185-192
1982
Homo sapiens
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brenda
Dahlmann, N.
A one-step liquid-chromatographic technique for the estimation of the deoxythymidine-5-triphosphatase in human serum
Hoppe-Seyler's Z. Physiol. Chem.
365
1263-1269
1984
Homo sapiens
brenda
Dahlmann, N.
Human serum thymidine triphosphate nucleotidohydrolase: purification and properties of a new enzyme
Biochemistry
21
6634-6639
1982
Homo sapiens
brenda
Dunham, L.F.; Price, A.R.
Deoxythymidine triphosphate-deoxyuridine triphosphate nucleotidohydrolase induced by Bacillus subtilis bacteriophage phie
Biochemistry
13
2667-2672
1974
Bacillus subtilis
brenda
Price, A.R.; Dunham, L.F.; Walker, R.L.
Thymidine triphosphate nucleotidohydrolase and desoxyuridylate hydroxymethylase induced by mutants of Bacillus subtilis bacteriophage SP82G
J. Virol.
10
1240-1241
1972
Bacillus subtilis
brenda
Dahlmann, N.
Serum desoxythymidine-5'-triphosphatase activity in lymphoproliferative disorders of men and mice
Anticancer Res.
11
225-228
1991
Homo sapiens, Mus musculus
brenda
Schultes, B.C.; Fischbach, E.; Dahlmann, N.
Purification and characterization of two different thymidine-5'-triphosphate-hydrolysing enzymes in human serum
Biol. Chem. Hoppe-Seyler
373
237-247
1992
Homo sapiens
brenda
Schultes, B.C.; Dahlmann, N.
Homogenenous preparation of human thymidine-5'-triphosphatase by electroelution from SDS/PAGE with subsequent renaturation
Eur. J. Biochem.
192
201-205
1990
Homo sapiens
brenda
Crampton, D.J.; Mukherjee, S.; Richardson, C.C.
DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase
Mol. Cell
21
165-174
2006
Escherichia phage T7
brenda
Donmez, I.; Patel, S.S.
Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase
EMBO J.
27
1718-1726
2008
Escherichia phage T7
brenda
Satapathy, A.K.; Richardson, C.C.
The glutamate switch of bacteriophage T7 DNA helicase: role in coupling nucleotide triphosphate (NTP) and DNA binding to NTP hydrolysis
J. Biol. Chem.
286
23113-23120
2011
Escherichia phage T7
brenda
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