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Information on EC 2.7.7.7 - DNA-directed DNA polymerase and Organism(s) Thermus aquaticus and UniProt Accession Q9XDH5

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EC Tree
     2 Transferases
         2.7 Transferring phosphorus-containing groups
             2.7.7 Nucleotidyltransferases
                2.7.7.7 DNA-directed DNA polymerase
IUBMB Comments
Catalyses DNA-template-directed extension of the 3'- end of a DNA strand by one nucleotide at a time. Cannot initiate a chain de novo. Requires a primer, which may be DNA or RNA. See also EC 2.7.7.49 RNA-directed DNA polymerase.
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This record set is specific for:
Thermus aquaticus
UNIPROT: Q9XDH5
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Word Map
The taxonomic range for the selected organisms is: Thermus aquaticus
The enzyme appears in selected viruses and cellular organisms
Synonyms
dna polymerase alpha, dna polymerase beta, dna polymerase iii, pol beta, klenow fragment, dna polymerase delta, taq dna polymerase, pol delta, pol alpha, dna polymerase gamma, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DNA polymerase III
-
deoxynucleate polymerase
-
-
-
-
deoxyribonucleate nucleotidyltransferase
-
-
-
-
deoxyribonucleic acid duplicase
-
-
-
-
deoxyribonucleic acid polymerase
-
-
-
-
deoxyribonucleic duplicase
-
-
-
-
deoxyribonucleic polymerase
-
-
-
-
deoxyribonucleic polymerase I
-
-
-
-
DNA duplicase
-
-
-
-
DNA nucleotidyltransferase
-
-
-
-
DNA nucleotidyltransferase (DNA-directed)
-
-
-
-
DNA polmerase beta
-
-
-
-
DNA polymerase
DNA polymerase alpha
-
-
-
-
DNA polymerase gamma
-
-
-
-
DNA polymerase I
DNA polymerase II
-
-
-
-
DNA polymerase III
-
-
-
-
DNA replicase
-
-
-
-
DNA-dependent DNA polymerase
-
-
-
-
duplicase
-
-
-
-
Klenow fragment
-
-
-
-
nucleotidyltransferase, deoxyribonucleate
-
-
-
-
Pol gamma
-
-
-
-
reverse transcriptase
-
-
sequenase
-
-
-
-
Taq DNA polymerase
Taq Pol I
-
-
-
-
Taq polymerase
Tca DNA polymerase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
show the reaction diagram
binding mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (DNA-directed)
Catalyses DNA-template-directed extension of the 3'- end of a DNA strand by one nucleotide at a time. Cannot initiate a chain de novo. Requires a primer, which may be DNA or RNA. See also EC 2.7.7.49 RNA-directed DNA polymerase.
CAS REGISTRY NUMBER
COMMENTARY hide
9012-90-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
dADP + DNAn
phosphate + DNAn+1
show the reaction diagram
activation energy analysis of the forward (DNA synthesis) and reverse (phosphorolysis of DNA) reactions catalyzed by the Taq DNA polymerase shows that DNA synthesis is strongly favored, allowing robust replication from low-energy substrates
-
-
?
dATP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
-
?
dCTP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
dTTP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
the mutant enzyme shows single deoxynucleotide additions with dCTP, dATP and dTTP, but not with dGTP as it results in addition of two successive base incorporations on the chosen template 2 hybridised to the DNA primer 1, thereby invalidating the single-turnover kinetic model, Michaelis-Menten mechanism, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
DNA replication can be accomplished using dNDPs as substrates. In thermophiles, genome replication may be less sensitive to the energy charge of the cell than in mesophiles because thermostable polymerases can accept the diphosphorylated as well as the triphosphorylated substrates. DNA replication is thus less affected by the intracellular ATP/ADP ratio, and the relatively high efficiency with which DNA is synthesized at elevated temperatures suggests that thermophiles may be able to dispense with the triphosphorylated substrates entirely
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aptamer
-
nucleotide ligands of small size and unique three-dimensional structure, TQ21 family, presence of loop is necessary for inhibition
-
catalpol
-
competitive
pyranicin
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Polyethylene glycol
-
4–10% of PEG 4000 or PEG 8000 can enhance the activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.022
dATP
-
pH 9.0, 37°C, recombinant mutant enzyme
0.024
dCTP
-
pH 9.0, 37°C, recombinant mutant enzyme
0.14
dTTP
-
pH 9.0, 37°C, recombinant mutant enzyme
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6
dADP
pH and temperature not specified in the publication
0.96
dATP
-
pH 9.0, 37°C, recombinant mutant enzyme
0.89
dCTP
-
pH 9.0, 37°C, recombinant mutant enzyme
0.0667 - 0.15
deoxynucleoside triphosphate
1.4
dTTP
-
pH 9.0, 37°C, recombinant mutant enzyme
additional information
additional information
the average speed of utilization of dNTPs by the Taq polymerase is kcat: 47/s
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
44
dATP
-
pH 9.0, 37°C, recombinant mutant enzyme
37
dCTP
-
pH 9.0, 37°C, recombinant mutant enzyme
9.9
dTTP
-
pH 9.0, 37°C, recombinant mutant enzyme
additional information
additional information
DNA synthesis from dNDPs is a little over an order of magnitude lower than from dNTPs and that Vmax/KM is about 400 times lower for dNDPs than for dNTPs
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
pyranicin
Thermus aquaticus
-
IC50 above 0.2 mM, DNA polymerase I, in 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 50% (v/v) glycerol, 0.1 mM EDTA, at 74°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.5
-
polymerase I
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
70
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Uniprot
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
DNA replication can be accomplished using dNDPs as substrates. In thermophiles, genome replication may be less sensitive to the energy charge of the cell than in mesophiles because thermostable polymerases can accept the diphosphorylated as well as the triphosphorylated substrates. DNA replication is thus less affected by the intracellular ATP/ADP ratio, and the relatively high efficiency with which DNA is synthesized at elevated temperatures suggests that thermophiles may be able to dispense with the triphosphorylated substrates entirely
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
DPO3A_THEAQ
1220
0
137390
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
62865
-
x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry
62879
-
x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry
94000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion method, crystal structure of Thermus aquaticus DNA polymerase III alpha subunit reveals that the structure of the catalytic domain of the eubacterial replicative polymerase is unrelated to that of the eukaryotic replicative polymerase but rather belongs to the Polbeta-like nucleotidyltransferase superfamily
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E602V/A608V/I614M/E615G
the mutant enzyme is able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wild-type enzyme incorporates dNTPs. The mutant enzyme allowed the generation of mixed RNA–DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. The mutant displays an expanded substrate spectrum towards other 2'-substituted nucleotides and is able to synthesize nucleic acid polymers in which each base bear a different 2'-substituent
G418K
-
increased exonuclease activity
L831N/A814R
truncated enzyme delta413-L813N/A814R has reduced temperature stability
N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R /S612N/V730L/R736Q/S739N/M747R
-
selection of a polymerase with 15 mutations, mostly located at the template binding interface, by directed evolution of Thermus aquaticus DNA polymerase I, the mutant enzyme is a single variant of the Stoffel fragment of Taq DNA polymerase I, the enzyme shows broad template specificity and is a thermostable DNA-dependent and RNA-dependent DNA-polymerase, see also EC 2.7.7.49
Q507E
-
increased exonuclease activity
additional information
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100
-
half-life at 3 MPa is 5.2 min, half-life at 45 MPa is 13 min, half-life at 89 MPa is 39 min
80
71% wild-type enzyme activity after 5 min, 8% delta413 mutant enzyme activity remains after 5 min, 32% delta422 mutant enzyme activity after 5 min, 50% delta413-L813N/A814R mutant enzyme activity after 5 min
95
61% wild-type enzyme activity after 5 min, no delta413 mutant enzyme activity remains after 5 min, 11% delta422 mutant enzyme activity after 5 min, 20% delta413-L813N/A814R mutant enzyme activity after 5 min
97.5
-
half-life: 9 min
additional information
-
the enzyme is stabilized in vitro by hydrostatic pressure at denaturing temperature of 100°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is stabilized in vitro by hydrostatic pressure at denaturing temperature of 100°C
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, stable over 12 months -70°C, stable over 18 months
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
high-level expression
-
polymerase I, expressed in Escherichia coli
-
recombinant enzyme, one-step purification on mABSGu adsorbent (immobilized ligand of the general formula X–Trz–Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz))
-
recombinant mutant DNA polymerase I from Escherichia coli strain BL21 AI cytoplasm by metal ion and heparin affinity chromatography
-
strain INValphaF' of Escherichia coli transformed with the pTaq plasmid containing the Taq gene expressed under control of the tac promoter
-
the purified recombinant Taq DNA polymerase shows better performance in comparison to commercially available polymerases
truncated enzyme
wild-type and mutant enzymes
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of chimeric enzymes in Escherichia coli
expression of mutant DNA polymerase I in Escherichia coli strain BL21 AI cytoplasm
-
high-level expression in Escherichia coli BL21. A 2500 base pair Taq DNA polymerase gene is colned into pGEX-4T-1 vector, containing a GSTtag, downstream of tac promoter and overexpressed it using isopropyl ß-d-1-thiogalactopyranoside (IPTG) as an inducer
polymerase I gene expressed in Escherichia coli
-
truncated versions lacking 288,422 and 413 amino acids at the N-terminus, fusion protein with beta-galactosidase
wild-type and mutant enzymes
-
wild-type and truncated form
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
diagnostics
-
a mutated thermostable DNA polymerase, Taq M1, from Thermus aquaticus, that exhibits an increased reverse transcriptase activity, is therefore designated for one-step PCR pathogen detection using established real-time detection methods
molecular biology
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pluthero, F.G.
Rapid purification of high-activity Taq DNA polymerase
Nucleic Acids Res.
21
4850-4851
1993
Thermus aquaticus, Thermus aquaticus INValphaF'
Manually annotated by BRENDA team
Campos, L.R.; Suarez, M.; Vazquez, A.; de la Fuente, R.; Herrera, L.
Molecular cloning of the gene, expression in E. coli and purification of the Thermus aquaticus DNA polymerase I
Acta Biotechnol.
12
155-159
1992
Thermus aquaticus, Thermus aquaticus YT1
-
Manually annotated by BRENDA team
Lawyer, F.C.; Stoffel, S.; Saiki, R.K.; Chang, S.Y.; Landre, P.A.; Abramson, R.D.; Gelfand, D.H.
High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5 to 3 exonuclease activity
PCR Methods Appl.
2
275-287
1993
Thermus aquaticus
Manually annotated by BRENDA team
Ma, W.P.; Kaiser, M.W.; Lyamicheva, N.; Schaefer, J.J.; Allawi, H.T.; Takova, T.; Neri, B.P.; Lyamichev, V.I.
RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases
J. Biol. Chem.
275
24693-24700
2000
Thermus thermophilus, Thermus aquaticus
Manually annotated by BRENDA team
Villbrandt, B.; Sagner, G.; Schomburg, D.
Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments
Protein Eng.
10
1281-1288
1997
Thermus aquaticus (P19821), Thermus aquaticus
Manually annotated by BRENDA team
Yakimovich, O.Y.; Alekseev, Y.I.; Maksimenko, A.V.; Voronina, O.L.; Lunin, V.G.
Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase
Biochemistry (Moscow)
68
228-235
2003
Thermus aquaticus
Manually annotated by BRENDA team
Pungitore, C.R.; Juri Ayub, M.; Borkowski, E.J.; Tonn, C.E.; Ciuffo, G.M.
Inhibition of Taq DNA polymerase by catalpol
Cell. Mol. Biol.
50
767-772
2004
Thermus aquaticus
Manually annotated by BRENDA team
Sasaki, Y.; Miyoshi, D.; Sugimoto, N.
Effect of molecular crowding on DNA polymerase activity
Biotechnol. J.
1
440-446
2006
Tequatrovirus T4, Thermus aquaticus
Manually annotated by BRENDA team
Melissis, S.; Labrou, N.E.; Clonis, Y.D.
One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography
Biotechnol. J.
2
121-132
2007
Thermus aquaticus
Manually annotated by BRENDA team
Bailey, S.; Wing, R.A.; Steitz, T.A.
The structure of T. aquaticus DNA polymerase III is distinct from eukaryotic replicative DNA polymerases
Cell
126
893-904
2006
Thermus aquaticus (Q9XDH5), Thermus aquaticus
Manually annotated by BRENDA team
Ong, J.L.; Loakes, D.; Jaroslawski, S.; Too, K.; Holliger, P.
Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide
J. Mol. Biol.
361
537-550
2006
Thermus aquaticus (P19821)
Manually annotated by BRENDA team
Naganuma, M.; Nishida, M.; Kuramochi, K.; Sugawara, F.; Yoshida, H.; Mizushina, Y.
1-deoxyrubralactone, a novel specific inhibitor of families X and Y of eukaryotic DNA polymerases from a fungal strain derived from sea algae
Bioorg. Med. Chem.
16
2939-2944
2008
Tequatrovirus T4, Bos taurus, Brassica oleracea, Escherichia coli, Homo sapiens, Rattus norvegicus, Thermus aquaticus
Manually annotated by BRENDA team
Angileri, F.F.; Aguennouz, M.; Conti, A.; La Torre, D.; Cardali, S.; Crupi, R.; Tomasello, C.; Germano, A.; Vita, G.; Tomasello, F.
Nuclear factor-kappaB activation and differential expression of survivin and Bcl-2 in human grade 2-4 astrocytomas
Cancer
112
2258-2266
2008
Thermus aquaticus
Manually annotated by BRENDA team
Nishida, M.; Hada, T.; Kuramochi, K.; Yoshida, H.; Yonezawa, Y.; Kuriyama, I.; Sugawara, F.; Yoshida, H.; Mizushina, Y.
Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.)
Food Chem.
108
551-560
2008
Tequatrovirus T4, Bos taurus, Brassica oleracea, Drosophila melanogaster, Escherichia coli, Homo sapiens, Rattus norvegicus, Thermus aquaticus, Oncorhynchus masou
Manually annotated by BRENDA team
Takahashi, S.; Yonezawa, Y.; Kubota, A.; Ogawa, N.; Maeda, K.; Koshino, H.; Nakata, T.; Yoshida, H.; Mizushina, Y.
Pyranicin, a non-classical annonaceous acetogenin, is a potent inhibitor of DNA polymerase, topoisomerase and human cancer cell growth
Int. J. Oncol.
32
451-458
2008
Tequatrovirus T4, Bos taurus, Brassica oleracea, Drosophila melanogaster, Escherichia coli, Homo sapiens, Rattus norvegicus, Thermus aquaticus, Oncorhynchus masou
Manually annotated by BRENDA team
Berdis, A.
Mechanisms of DNA polymerases
Chem. Rev.
109
2862-2879
2009
Geobacillus stearothermophilus, Bacillus subtilis, Tequatrovirus T4, Escherichia phage T7, Escherichia coli, Homo sapiens, Escherichia phage RB69, Thermus aquaticus (P19821)
Manually annotated by BRENDA team
Kranaster, R.; Drum, M.; Engel, N.; Weidmann, M.; Hufert, F.T.; Marx, A.
One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase
Biotechnol. J.
5
224-231
2010
Thermus aquaticus
Manually annotated by BRENDA team
Vichier-Guerre, S.; Jestin, J.L.
Characterisation of a DNA polymerase highly mutated along the template binding interface
Mol. Biotechnol.
46
58-62
2010
Thermus aquaticus
Manually annotated by BRENDA team
Summit, M.; Scott, B.; Nielson, K.; Mathur, E.; Baross, J.
Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms
Extremophiles
2
339-345
1998
Pyrococcus sp., Pyrococcus furiosus, Thermus aquaticus, Pyrococcus sp. ES4
Manually annotated by BRENDA team
Villbrandt, B.; Sobek, H.; Frey, B.; Schomburg, D.
Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase
Protein Eng.
13
645-654
2000
Thermus aquaticus (P19821), Thermus aquaticus
Manually annotated by BRENDA team
Din, R.U.; Khan, M.I.; Jan, A.; Khan, S.A.; Ali, I.
A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli
Arch. Microbiol.
202
1449-1458
2020
Thermus aquaticus (P19821), Thermus aquaticus
Manually annotated by BRENDA team
Burke, C.R.; Luptak, A.
DNA synthesis from diphosphate substrates by DNA polymerases
Proc. Natl. Acad. Sci. USA
115
980-985
2018
Thermus aquaticus (P19821), Thermococcus litoralis (P30317), Geobacillus stearothermophilus (P52026), Pyrococcus furiosus (P61875), Bacillus subtilis (P94544), Bacillus subtilis 168 (P94544)
Manually annotated by BRENDA team