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Information on EC 2.7.7.7 - DNA-directed DNA polymerase and Organism(s) Pyrococcus furiosus and UniProt Accession P61875

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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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Select one or more organisms in this record: ?
This record set is specific for:
Pyrococcus furiosus
UNIPROT: P61875
Word Map
The taxonomic range for the selected organisms is: Pyrococcus furiosus
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
deoxynucleate polymerase
-
-
-
0
deoxyribonucleate nucleotidyltransferase
-
-
-
0
deoxyribonucleic acid duplicase
-
-
-
0
deoxyribonucleic acid polymerase
-
-
-
0
deoxyribonucleic duplicase
-
-
-
0
deoxyribonucleic polymerase
-
-
-
0
deoxyribonucleic polymerase I
-
-
-
0
DNA duplicase
-
-
-
0
DNA nucleotidyltransferase
-
-
-
0
DNA nucleotidyltransferase (DNA-directed)
-
-
-
0
DNA polmerase beta
-
-
-
0
DNA polymerase
DNA polymerase alpha
-
-
-
0
DNA polymerase gamma
-
-
-
0
DNA polymerase I
DNA polymerase II
-
-
-
0
DNA polymerase III
-
-
-
0
DNA replicase
-
-
-
0
DNA-dependent DNA polymerase
-
-
-
0
duplicase
-
-
-
0
Klenow fragment
-
-
-
0
nucleotidyltransferase, deoxyribonucleate
-
-
-
0
Pfu
296986
a family B DNA polymerase
Pfu Pol
321
-
Pfu-POl
321
-
Pol gamma
-
-
-
0
Pol II
299070
-
replicative DNA polymerase
321
-
sequenase
-
-
-
0
Taq DNA polymerase
-
-
-
0
Taq Pol I
-
-
-
0
Tca DNA polymerase
-
-
-
0
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
-
-
-
0
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
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
dATP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
additional information
?
-
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
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
optimum: 25-150 mM
MgCl2
optimal concentration: 10 mM
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ddTTP
0.4 mM, about 80% loss of activity
hypoxanthine
-
traces of uracil and hypoxanthine in DNA can lead to inhibition of the polymerase chain reaction by archaeal DNA polymerases
N-ethylmaleimide
1 mM, about 80% loss of activity
Uracil
-
traces of uracil and hypoxanthine in DNA can lead to inhibition of the polymerase chain reaction by archaeal DNA polymerases
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 0.13
dATP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.022 - 0.32
dATP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 14
dATP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.07
75°C, pH not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.8
pH 7.0: about 75% of maximal activity, pH 8.8: about 75% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
72
-
assay at
80
substrate: calf thymus activated DNA
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70 - 85
70°C: about 55% of maximal activity, 85°C: about 70% of maximal activity
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
replicative DNA polymerase
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
130000
gel filtration
218000
gel filtration
69294
x * 69294 + x + 143161, calculated from sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop method, structure of the apo form a binary complex of the archael B family DNA polymerase E10 variant with duplex DNA bound in synthesis mode
using 0.2 M ammonium sulfate, 0.1 M Na-cacodylate (pH 6.5), 5 mM dithiothreitol, 50 mM MnCl2, and 15% (w/v) PEG 8000
collection of diffraction data to 3.1 A of the selenomethionine-derivatized crystal, the crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90°, beta = 109.7°, and gamma = 90°
-
hanging-drop vapour diffusion method. Crystallization of a stable complex of the DNA polymerase with proliferating cell nuclear antigen (PCNA), using a PCNA monomer mutant. The best ordered crystal diffracts to 3.0 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3 A, b = 123.3 A, c = 91.3 A
-
the enzyme is crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals have unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffract beyond 4 A on a 1.08 A synchrotron radiation source
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A408S
-
A408S mutation results in a significant increase in both dNTP binding affinity and fidelity, kcat/Km for dATP is about 45% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity
D215A
-
3'-5'-exonuclease inactive mutant enzyme
D405A
-
mutant enzyme loses 99.8% of DNA polymerizing activity and 90% of 3'->5' exonucleolytic activity
D405E
-
mutant enzyme loses 95.8% of DNA polymerizing activity and 90% of 3'->5' exonucleolytic activity
D473G
-
wild-type variant Pfu-Pol(exo-) is is 60fold less accurate than Pfu-Pol(exo+)
DELTAH672-S775
-
mutant enzyme loses 99% of DNA polymerizing activity and 97% of 3'->5' exonucleolytic activity
DELTAL717-S775
-
mutant enzyme loses 97% of DNA polymerizing activity and 97% of 3'->5' exonucleolytic activity
DELTAL746-S775
-
mutant protein has DNA polymerizing activity with 2.3fold higher specific activity than that of the wild-type but retains only 10% of the 3'->5' exonucleolytic activity of the wild-type
L409I
-
kcat/Km for dATP is about 20% compared to the wild-type enzyme
L409M
-
L409 mutation results in drastically reduced affinity for the correct dNTP, a much higher efficiency of both misincorporation and mismatch extension, and substantially lower fidelity as demonstrated by a PCR-based forward mutation assay, kcat/Km for dATP is about 155% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity
L409V
-
kcat/Km for dATP is about 35% compared to the wild-type enzyme
T471A
-
less accurate, by factors of 1.6 than the wild-type variant Pfu-Pol(exo-)
T471G
-
less accurate, by factors of 1.2 than the wild-type variant Pfu-Pol(exo-)
Y410I
-
kcat/Km for dATP is about 20% compared to the wild-type enzyme
Y410L
-
kcat/Km for dATP is about 45% compared to the wild-type enzyme
Y410V
-
Y410V mutation results in high fidelity in both misincorporation assays and forward mutation assays, but displays a substantially higher Km than wild-type enzyme, kcat/Km for dATP is about 10% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
107.5
-
half-life at 3 MPa is 6.9 min, half-life at 45 MPa is 36 min, half-life at 89 MPa is 50 min
80
more than 80% of the initial activity is retained after incubation for 30 min
90
60 min, about 50% loss of activity
additional information
-
the enzyme is stabilized in vitro by hydrostatic pressure at denaturing temperature of 107.5°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is stabilized in vitro by hydrostatic pressure at denaturing temperature of 107.5°C
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE Sephacel gel filtration and HiTrap heparin column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
expression of Pfu in Escherichia by the expression plasmid pETpfu is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However the plasmid is stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90000 Da protein is expressed upon addition of isopropyl beta-D-thiogalactopyranoside
baculovirus-mediated expression in Spodoptera frugiperda (Sf9) cell, human placental alkaline phosphatase signal sequence (MLGPCMLLLLLLLGLRLQLSLG) proves to be optimal for the secretion
-
expression in Escherichia coli
overexpressed in Escherichia coli
-
overexpression in Escherichia coli from pET28a plasmids
-
the DNA polymerase from the archaebacterium Pyrococcus furiosus does not testify for a specific relationship between archaebacteria and eukaryotes
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Biles, B.D.; Connolly, B.A.
Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR
Nucleic Acids Res.
32
e176
2004
Pyrococcus furiosus
Manually annotated by BRENDA team
Nishida, H.; Matsumiya, S.; Tsuchiya, D.; Ishino, Y.; Morikawa, K.
Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus
Acta Crystallogr. Sect. F
62
253-256
2006
Pyrococcus furiosus
Manually annotated by BRENDA team
Kim, S.W.; Kim, D.U.; Kim, J.K.; Kang, L.W.; Cho, H.S.
Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus
Int. J. Biol. Macromol.
42
356-361
2008
Pyrococcus furiosus (P61875)
Manually annotated by BRENDA team
Goldman, S.; Kim, R.; Hung, L.W.; Jancarik, J.; Kim, S.H.
Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase
Acta Crystallogr. Sect. D
54
986-988
1989
Pyrococcus furiosus
Manually annotated by BRENDA team
Kennedy, E.M.; Hergott, C.; Dewhurst, S.; Kim, B.
The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate
Biochemistry
48
11161-11168
2009
Pyrococcus furiosus
Manually annotated by BRENDA team
Imamura, M.; Uemori, T.; Kato, I.; Ishino, Y.
A non-alpha-like DNA polymerase from the hyperthermophilic archaeon Pyrococcus furiosus
Biol. Pharm. Bull.
18
1647-1652
1995
Pyrococcus furiosus (P81409)
Manually annotated by BRENDA team
Uemori, T.; Sato, Y.; Kato, I.; Doi, H.; Ishino, Y.
A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization
Genes Cells
2
499-512
1997
Pyrococcus furiosus (P81412 and P81409), Pyrococcus furiosus
Manually annotated by BRENDA team
Gill, S.; ONeill, R.; Lewis, R.J.; Connolly, B.A.
Interaction of the family-B DNA polymerase from the archaeon Pyrococcus furiosus with deaminated bases
J. Mol. Biol.
372
855-863
2007
Pyrococcus furiosus
Manually annotated by BRENDA team
Forterre, P.
The DNA polymerase from the archaebacterium Pyrococcus furiosus does not testify for a specific relationship between archaebacteria and eukaryotes
Nucleic Acids Res.
20
1811
1992
Pyrococcus furiosus
Manually annotated by BRENDA team
Komori, K.; Ishino, Y.
Functional interdependence of DNA polymerizing and 3-->5 exonucleolytic activities in Pyrococcus furiosus DNA polymerase I
Protein Eng.
13
41-47
2000
Pyrococcus furiosus
Manually annotated by BRENDA team
Lu, C.; Erickson, H.P.
Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus
Protein Expr. Purif.
11
179-184
1997
Pyrococcus furiosus (P61875)
Manually annotated by BRENDA team
Nishida, H.; Tanabe, M.; Ishino, Y.; Oyama, T.; Morikawa, K.
Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus
Protein Pept. Lett.
14
403-405
2007
Pyrococcus furiosus
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
Mroczkowski, B.S.; Huvar, A.; Lernhardt, W.; Misono, K.; Nielson, K.; Scott, B.
Secretion of thermostable DNA polymerase using a novel baculovirus vector
J. Biol. Chem.
269
13522-13528
1994
Pyrococcus furiosus
Manually annotated by BRENDA team
Wynne, S.A.; Pinheiro, V.B.; Holliger, P.; Leslie, A.G.
Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA
PLoS One
8
e70892
2013
Pyrococcus furiosus (P61875)
Manually annotated by BRENDA team
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