Any feedback?
Information on EC 2.7.7.7 - DNA-directed DNA polymerase and Organism(s) Pyrococcus furiosus and UniProt Accession P61875
for references in articles please use BRENDA:EC2.7.7.7Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
2.7.7.7 DNA-directed DNA polymeraseIUBMB 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.
Specify your search results
Word Map
- 2.7.7.7
- strand
-
single-stranded
-
fidelity
-
holoenzyme
- mismatch
- bacteriophage
- pcna
- fork
- polymerization
- aphidicolin
- duplex
-
proofreading
-
calf
- endonuclease
- thymus
-
clamp
-
misincorporation
- helicase
-
abasic
-
template-primer
- 5'-triphosphate
- dntps
-
stall
- thymine
-
error-free
- mispairs
-
anneal
-
sliding
-
uv-induced
-
incoming
-
exonucleolytic
- replisome
-
transversions
- deoxyribonucleoside
- xeroderma
- pigmentosum
- ssdna
-
deoxynucleotide
-
hypermutation
-
undamaged
-
okazaki
- dtmp
-
slippage
-
cyclobutane
- transcriptases
- dnab
-
watson-crick
-
high-fidelity
-
myeloblastosis
- aquaticus
- synthesis
- analysis
- drug development
- diagnostics
- biotechnology
- medicine
- molecular biology
The taxonomic range for the selected organisms is: Pyrococcus furiosus
The enzyme appears in selected viruses and cellular organisms
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
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
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
-
-
-
-
sequenase
-
-
-
-
Taq DNA polymerase
-
-
-
-
Taq Pol I
-
-
-
-
Tca DNA polymerase
-
-
-
-
DNA polymerase I
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
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
9012-90-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn
diphosphate + DNAn+1
-
-
-
?
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn
diphosphate + DNAn+1
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
the DNA polymerase from Pyrococcus furiosus has the lowest error rate of any known polymerase in polymerase chain reaction amplification
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
wild-type Pfu-Pol makes about one mistake for every 1000000 bases incorporated, wild-type variant Pfu-Pol(exo-)(D473F)is 60fold less accurate
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme has a template-primer preference which is characteristic of a replicative DNA polymerase
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
-
measurement of the incorporation of methyl-TTP into acid insoluble material. The single-stranded DNA substrate is more sensitive than the double stranded substrate. The polymerase and exonuclease domains in the family B DNA polymerases are functionally interdependent
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
replicative DNA polymerase
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
the enzyme utilizes activated DNA as a template-primer, artificial substrate (activated poly(dA-dT)) is preferred by the enzyme. M13 single-stranded DNA primed with 17 base oligonucleotide is not a good substrate. DNA elongation ability of Pol II using a natural DNA template is much lower than that of Pol I from this organism and DNA synthesis of Pol II seems to be non-processive
-
-
?
additional information
?
-
-
polymerase binds DNA containing uracil 1.54.5-fold more strongly than hypoxanthine
-
-
?
additional information
?
-
the enzyme has strong 3'->5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase
-
-
?
additional information
?
-
-
the enzyme has strong 3'->5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn
diphosphate + DNAn+1
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
the enzyme has a template-primer preference which is characteristic of a replicative DNA polymerase
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
hypoxanthine
-
traces of uracil and hypoxanthine in DNA can lead to inhibition of the polymerase chain reaction by archaeal DNA polymerases
Uracil
-
traces of uracil and hypoxanthine in DNA can lead to inhibition of the polymerase chain reaction by archaeal DNA polymerases
additional information
DNA polymerizing activity is not inhibited by aphidicolin at concentrations up to 2 mM that inhibit the activities of Pfu Pol I and other alpha-like DNA polymerases.
-
additional information
-
DNA polymerizing activity is not inhibited by aphidicolin at concentrations up to 2 mM that inhibit the activities of Pfu Pol I and other alpha-like DNA polymerases.
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.002
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409M
0.0025
dATP
-
55°C, pH not specified in the publication, wild-type enzyme
0.01
dATP
-
55°C, pH not specified in the publication, mutant enzyme A408S
0.012
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409V
0.014
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409I
0.046
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410V
0.069
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410L
0.13
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410I
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.022
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409I
0.028
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409M
0.029
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410V
0.034
dATP
-
55°C, pH not specified in the publication, mutant enzyme L409V
0.037
dATP
-
55°C, pH not specified in the publication, mutant enzyme A408S
0.31
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410L
0.32
dATP
-
55°C, pH not specified in the publication, mutant enzyme Y410I
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
70 - 85
70°C: about 55% of maximal activity, 85°C: about 70% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
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
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
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
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
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
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
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-)
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
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
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
purified with the AKTAprime plus compact one-step purification system by Ni2+ chelating affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
recombinant plasmid pLysS His6-tagged Pfu-pET28a is constructed. His-tagged Pfu is expressed in Escherichia coli BL21 (DE3) competent cells
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
the DNA polymerase from the archaebacterium Pyrococcus furiosus does not testify for a specific relationship between archaebacteria and eukaryotes
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
DNA polymerase plays prominent roles in numerous biotechnologies. The use of diphosphate substrates has the potential to make practical the incorporation of expensive analogs, such as isotopically labeled or chemically modified nucleotides, eliminating the need for challenging triphosphate syntheses. This feature of DNA polymerases may also provide a method for detecting nucleotides used in high-throughput DNA sequencing
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
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
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), Pyrococcus furiosus
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
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
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)
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
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
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
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
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)
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
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
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
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)
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)
EXTERNAL LINKS (specific for EC-Number 2.7.7.7)
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
