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Information on EC 2.7.7.7 - DNA-directed DNA polymerase and Organism(s) Saccharolobus solfataricus and UniProt Accession P26811

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     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:
Saccharolobus solfataricus
UNIPROT: P26811
Word Map
The taxonomic range for the selected organisms is: Saccharolobus solfataricus
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
B-family replicative DNA polymerase
2070
-
Dbh DNA polymerase
2070, 297830
-
Dbh polymerase
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 4
286134
-
DNA polymerase alpha
-
-
-
0
DNA polymerase B1
DNA polymerase Dpo4
DNA polymerase gamma
-
-
-
0
DNA polymerase I
-
-
-
0
DNA polymerase II
-
-
-
0
DNA polymerase III
DNA polymerase IV
DNA replicase
-
-
-
0
DNA replication polymerase
2070
-
DNA-dependent DNA polymerase
duplicase
-
-
-
0
error-prone DNA polymerase
297830
-
Klenow fragment
-
-
-
0
lesion-bypass DNA polymerase
286134
-
nucleotidyltransferase, deoxyribonucleate
-
-
-
0
Pol gamma
-
-
-
0
sequenase
-
-
-
0
Sso DNA pol B1
2070
-
Sso DNA pol Y1
2070
-
Sso DNA polymerase Y1
2070
-
Sso DNApol
297286
-
SSO0552
297286
locus name
SSO2448
Taq DNA polymerase
-
-
-
0
Taq Pol I
-
-
-
0
Tca DNA polymerase
-
-
-
0
translesion DNA polymerase
2070
-
translesion polymerase Dpo4
2070
-
additional information
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a 2'-deoxyribonucleoside 5'-triphosphate + DNAn = diphosphate + DNAn+1
show the reaction diagram
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
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate + DNAn
diphosphate + ?
show the reaction diagram
-
-
-
?
2-thio-dCTP + DNAn
?
show the reaction diagram
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
5-methyl-dCTP + DNAn
?
show the reaction diagram
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
7-deaza-2'-deoxyadenosine 5'-triphosphate + DNAn
diphosphate + ?
show the reaction diagram
-
-
-
?
8-bromo-dATP + DNAn
?
show the reaction diagram
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
8-oxo-dATP + DNAn
?
show the reaction diagram
dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine. dPTP (i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-b-d-2’-deoxyribofuranosid-5’-triphosphate) is incorporated opposite guanine slightly less efficiently than dCTP and is not incorporated opposite O6-methylguanine
-
-
?
dATP + DNAn
?
show the reaction diagram
dATP + DNAn
diphosphate + ?
show the reaction diagram
-
-
-
?
dATP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
dCTP + DNAn
?
show the reaction diagram
dCTP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
deoxynucleoside triphosphate + DNAn
?
show the reaction diagram
the enzyme can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene. An anti glycosidic torsion with C1'-exo deoxyribose conformation allows N-(deoxyguanosin-8-yl)-2-acetylaminofluorene to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting
-
-
?
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
dGTP + DNAn
?
show the reaction diagram
dGTP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
dITP + DNAn
?
show the reaction diagram
-
-
-
-
?
DNA 21/41-mer + dTTP
? + diphosphate
show the reaction diagram
kinetic mechanism for DNA polymerization is proposed, the enzyme utilizes an induced-fit mechanism to select correct incoming nucleotides
-
-
?
dPTP + DNAn
?
show the reaction diagram
i.e. 6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one-8-beta-D-2'-deoxyribofuranosid 5'-triphosphate. dCTP and 5-methyl-dCTP are efficiently incorporated opposite a template guanine but significantly less so opposite a template O6-methylguanine. 2-thio-dCTP is efficiently inserted opposite guanine and is also incorporated opposite O6-methylguanine, to a similar extent as dCTP. Of the dNTPs assayed, dCTP, 5-Me-dCTP, and 2-thio-dCTP display the highest incorporation efficiency opposite O6-methylguanine. dTTP incorporation is favored opposite O6-methylguanine rather than opposite guanine. Hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. 8-oxo-dATP and 8-bromo-dATP are not inserted opposite O6-methylguanine and are slowly incorporated opposite guanine
-
-
?
dTTP + DNAn
?
show the reaction diagram
dTTP + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
dNTP insertion opposite a benzo[a]pyrene-N2-dG-adduct
-
-
?
N1-methyl-2'-deoxyadenosine 5'-triphosphate + DNAn
diphosphate + ?
show the reaction diagram
-
-
-
?
North-methanocarba-dATP + DNAn
?
show the reaction diagram
South-methanocarba-dATP + DNAn
?
show the reaction diagram
the role of sugar geometry during nucleotide selection is probed by the enzyme from Sulfolobus solfataricus using fixed conformation nucleotide analogues. The enzyme relatively tolerant to the substrate conformation: North-methanocarba-dATP that locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker or South-methanocarba-dATP that locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker
-
-
?
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
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
DNA polymerase Dpo2 and Dpo3 are both more active with Mg2+ than Mn2+. DNA polymerase Dpo1 and Dpo4 are similarly active with Mg2+ or Mn2+
Mn2+
DNA polymerase Dpo2 and Dpo3 are both more active with Mg2+ than Mn2+. DNA polymerase Dpo1 and Dpo4 are similarly active with Mg2+ or Mn2+
MgCl2
optimal concentration: 5 mM
NaCl
optimal concentration: 0-75 mM
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-(2'-deoxy-beta-D-erythro-pentofuranosyl) pyrimido[1,2-alpha]purin-10(3H)-one
-
i.e. M1dG. When paired opposite cytosine in duplex DNA at physiological pH,M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG. To improve the understanding of the basis for M1dG-induced mutagenesis, the mechanism of translesion DNA synthesis opposite M1dG by the model Y-family polymerase Dpo4 is studied at a molecular level using kinetic and structural approaches. Steady-state and transient-state kinetic results both indicate that Dpo4 catalysis is inhibited by M1dG (260-2900-fold), with dATP being the favored insertion event for both sequences tested
cis-syn thymine dimer
Dpo4 is severely blocked by the presence of a cis-syn thymine dimer in the template
N2,N2-dimethyl guanine
the presence of N2,N2-dimethyl guanine lowers the catalytic efficiency for incorporation of dCTP of DNA polymerase Dpo4 16000fold
N2,N2-dimethylguanine
the presence of N2,N2-dimethylguanine lowers the catalytic efficiency of the DNA polymerase Dpo4 16000fold. Dpo4 inserts dNTPs almost at random during bypass of N2,N2-dimethylguanine, and much of the enzyme is kinetically trapped by an inactive ternary complex when N2,N2-dimethylguanine is present
Replication factor C
DNA polymerization by Dpo2 and Dpo3 is strongly inhibited
-
single-stranded binding protein
-
SsoCdc6-2 protein
-
inhibits both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it forms a large complex with SsoPolY and stimulates DNA-binding activity on paired primer-template DNA substrates
-
additional information
-
reverse gyrase inhibits DNA polymerase PolB1. Inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control DNA polymerase PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a mechanism of regulation of these enzymes
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
proliferating cell nuclear antigen
DNA synthesis by any of the DNA polymerases alone is not very processive. The addition of proliferating cell nuclear antigen slightly increases primer extension only by DNA polymerase Dpo4 but not by DNA polymerase Dpo1, Dpo2, or Dpo3. Both proliferating cell nuclear antigen and replication factor C enhance primer extension substantially in the cases of DNA polymerase Dpo1, Dpo3, and Dpo4 (which generate much longer extension products up to about 150-, 65-, and 150-mers, respectively) but very weakly with DNA polymerase Dpo2, which generates slightly more products of similar length up to about 80-mers. The addition of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein strongly increases DNA polymerization by Dpo1 and Dpo4, which yields extended products, mainly up to about 500-mers and 300-mers, respectively. An increase of the processivity of DNA synthesis in the presence of all accessory replication factors is the most prominent with Dpo1 and also with Dpo4
-
Replication factor C
both proliferating cell nuclear antigen and replication factor C enhance primer extension substantially in the cases of DNA polymerase Dpo1, Dpo3, and Dpo4 (which generate much longer extension products up to about 150-, 65-, and 150-mers, respectively) but very weakly with DNA polymerase Dpo2, which generates slightly more products of similar length up to about 80-mers. The addition of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein strongly increases DNA polymerization by Dpo1 and Dpo4, which yields extended products, mainly up to about 500-mers and 300-mers, respectively. An increase of the processivity of DNA synthesis in the presence of all accessory replication factors (proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein) is the most prominent with Dpo1 and also with Dpo4
-
single-stranded binding protein
an increase of the processivity of DNA synthesis in the presence of all accessory replication factors (proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein) is the most prominent with Dpo1 and also with Dpo4 as compared with DNA polymerase Dpo2 and Dpo3
-
proliferating cell nuclear antigen
-
Replication factor C
-
single-stranded binding protein
-
SsoCdc6
-
Orc1/Cdc6 proteins stimulate the DNA-binding ability of SsoPolB1 and differentially regulate both its polymerase and nuclease activities
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.1
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0084
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
2.5
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
3.1
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.006 - 0.0144
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
0.067 - 0.98
2-thio-dCTP
0.013 - 1.22
5-Methyl-dCTP
0.0011 - 0.344
7-deaza-2'-deoxyadenosine 5'-triphosphate
0.0008 - 2.2
dATP
0.000077 - 2.5
dCTP
0.012 - 1.2
dGTP
0.16
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.006 - 8.9
dTTP
0.223 - 0.403
N1-methyl-2'-deoxyadenosine 5'-triphosphate
0.0012 - 0.0087
North-methanocarba-dATP
0.0013
South-methanocarba-dATP
pH 7.5, 37°C
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0018
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
0.23
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0068
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.096
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.29 - 0.34
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
0.03 - 0.04
2-thio-dCTP
0.02 - 0.33
5-Methyl-dCTP
0.068 - 0.405
7-deaza-2'-deoxyadenosine 5'-triphosphate
0.000016 - 2.5
dATP
0.000053 - 12
dCTP
0.000013 - 3.7
dGTP
0.05
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.0000072 - 0.64
dTTP
0.012 - 0.15
N1-methyl-2'-deoxyadenosine 5'-triphosphate
0.014 - 0.08
North-methanocarba-dATP
0.0088
South-methanocarba-dATP
pH 7.5, 37°C
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00000086
dATP
pH 7.5, 37°C, DNA polymerase Dpo1
0.027
dCTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.0000027
dGTP
pH 7.5, 37°C, DNA polymerase Dpo1
0.000031
dTTP
pH 7.5, 37°C, DNA polymerase Dpo1
20.14 - 56.7
2-aminopurine-2'-deoxy-D-ribose 5'-triphosphate
0.03 - 0.6
2-thio-dCTP
0.016 - 25.38
5-Methyl-dCTP
0.2 - 240.9
7-deaza-2'-deoxyadenosine 5'-triphosphate
0.000000041 - 287.5
dATP
0.0000054 - 370
dCTP
0.000000068 - 32
dGTP
0.31
dPTP
pH 7.4, 37°C, steady-state kinetics for single nucleotide primer extension with guanine as template
0.0000000019 - 35
dTTP
0.04 - 0.625
N1-methyl-2'-deoxyadenosine 5'-triphosphate
9.2 - 11.7
North-methanocarba-dATP
6.8
South-methanocarba-dATP
pH 7.5, 37°C
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
70°C, pH is not specified in the publication
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
pH 7.0: about 50% of maximal activity, pH 9.0: about 60% of maximal activity
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 70
activities of DNA polymerase Dpo1 gradually increases from 25 to 60°C and is substantial even at 70°C, with40% of the activity at 60°C
2 - 80
-
the incorporation rate for correct nucleotides increases by 18900fold from 2°C to 56°C, the fidelity of Dpo4 at 80°C is similar to that determined at 56°C
25 - 60
25 - 70
activity of DNA polymerase Dpo4 gradually increases from 25 to 60°C and is substantial even at 70°C, with60% of the activity at 60°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
-
replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication, kinetics and conformational dynamics by single-molecule Förster resonance energy transfer techniques, overview. The replicative polymerase can bind to DNA in at least three conformations, corresponding to an open and closed conformation of the finger domain as well as a conformation with the DNA substrate bound to the exonuclease active site of PolB1. PolB1 can transition between these conformations without dissociating from a primer-template DNA substrate. The closed conformation is promoted by a matched incoming dNTP but not by a mismatched dNTP and that mismatches at the primer-template terminus lead to an increase in the binding of the DNA to the exonuclease site
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
101224
x * 101224, calculated from sequence
110000
monomer, gel filtration
100000
110000
-
gel filtration, glycerol gradient centrifugation
40189
x * 40189, calculated from sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 101224, calculated from sequence
oligomer
the enzyme can form a oligomeric complex on DNA
trimer
the enzyme is able to physically associate with itself to form a trimer. This complex is stabilized in the presence of DNA. Initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. These are specific polymerase-polymerase interactions and not just separate binding events along DNA. The presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form
?
x * 40189, calculated from sequence
monomer
oligomer
the enzyme can form a oligomeric complex on DNA
additional information
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method at 21°C using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source
X-ray structure, 2.4 A resolution
2.3 A resolution crystal structure of a catalytic fragment
2.8 A resolution crystal structure
bypass crystal structures of Dpo4:DNA(S-cdG):dCTP (error-free) and Dpo4:DNA(S-cdG):dTTP (error-prone) are catalytically incompetent. In Dpo4:DNA(S-cdG):dTTP structure, S-cdG induces a loop structure and causes an unusual 5'-template base clustering at the active site, providing the first structural evidence for the previously suggested template loop structure that can be induced by a cyclopurine lesion
crystal data and refinement parameters for the ternary (protein/DNA/GTP) complexes of Dpo4, The complexes are crystallized by sitting-drop vapor diffusion
-
crystal forms for the N249Y mutant enzyme are obtained at 22°C by the microbatch method
crystal structure of Dpo4 in complex with North-methanocarba-dATP opposite dT
crystal structure of ternary complex Dpo4/blunt-end DNA/dATP, crystals are produced by the hanging-drop method at 20°C
crystal structures of Dpo4 complexes with oligonucleotides are solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG
crystal structures of Dpo4 in complex with DNA duplexes containing the 2,4-difluorotoluene analog. The structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite 2,4-difluorotoluene and its inability to extend beyond a G:2,4-difluorotoluene pair
crystallization of an enzyme:DNA complex, sitting drop vapor diffusion method by mixing 0.001 mL of complex with 0.001 mL of a solution containing 50 mM Tris-HCl (pH 7.4 at 25°C) buffer, 12-20% polyethylene glycol 3350 (w/v), 100 mM Ca(OAc)2, and 2.5% glycerol (v/v)
crystallization of enzyme-DNA complexes
crystallization of mutant enzymes R332A and R332E in complex with DNA and dGTP (R332E(8-oxoG:A), R332E(8-oxoG:C), R332A(8-oxoG:A) and R332A(8-oxoG:C)). The R332E(8-oxoG:C) structure is crystallized by the hanging drop vapor diffusion technique, using a mixture of 14% polyethylene glycol 4000 (w/v), 0.1 M calcium acetate, and 20 mM HEPES (pH 7.3) as reservoir
crystals are generated by the vapour diffusion in hanging drops. Crystal structure of the full-length enzyme Dpo4 in complex with heterodimeric sliding clamp PCNA1–PCNA2 at 2.05 A resolution. Two hinges render the multidomain polymerase flexible conformations and orientations relative to PCNA. The enzyme binds specifically to PCNA1 on the conserved ligand binding site
crystals are grown at room temperature by hanging drop vapor diffusion
crystals are grown using the sitting drop vapor diffusion method (pH 7.4 , 25°C). The Dpo4 polymerase is cocrystallized with the aflatoxin B1-formamidopyrimidine-modified template and the structure is determined at 3.0 A resolution. The structures of the ternary complexes are determined at 2.9 and 2.7 A resolutions for the dATP and dTTP complexes, respectively
determination of crystal structures of a binary Mg2+-form Dpo4–DNA complex with 1,N2-etheno-dG in the template strand as well as of ternary Mg2+-form Dpo4–DNA–dCTP/dGTP complexes with 8-oxoG in the template strand. Crystals are grown using the sitting-drop vapor-diffusion method by mixing equal amounts of Dpo4–DNA complex solution and of a reservoir solution containing 12–20% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M magnesium acetate and 20 mMTris pH 7.5
Dpo4 in complex with DNA duplex containing N2,N2-dimethyl-substituted guanine-modified template, sitting drop vapor diffusion method, using 10-15% polyethylene glycol 3350 (w/v), 30 mM NaCl, 100 mM MgCl2, and 3% glycerol (v/v)
hanging drop method, crystals of the enzyme in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide are analyzed by Raman microscopy. 13C- and 15N-labeled d*CTP, or unlabeled dCTP, are soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ and Mn2+, nucleotide incorporation is detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, is observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that is possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction does not occur. Instead, a ternary protein/template/d*CTP complex is formed
hanging-drop vapor diffusion method at 20°C
hanging-drop vapor diffusion method at 20°C, crystal structures of the enzyme in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, solved at 1.7 A and 2.1 A resolution, respectively
hanging-drop vapour-diffusion method at 21°C using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution
sitting drop vapor diffusion method, crystallization of Dpo4/(6S,8R,11S)-trans-4-hydroxynonenal-dGuo modified 18-mer template primer DNA complexes
sitting drop vapor diffusion method, crystallization of Dpo4/DNA complexes. Crystal structures reveal wobble pairing between C and O6-BzG
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sitting drop, vapor-diffusion method with the reservoir solution containing 10–15% polyethylene glycol 3350 (w/v), 30 mm NaCl, 100 mm MgCl2, and 3% glycerol (v/v). One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine opposite template N2,N2-dimethylguanine in a post-insertion position shows dideoxycytosine folded back into the minor groove, as a catalytically incompetent complex. A second crystal has two unique orientations for the primer terminal dideoxycytosine as follows: (I) flipped into the minor groove and (II) a long pairing with N2,N2-dimethylguanine in which one hydrogen bond exists between the O-2 atom of dideoxycytosine and the N-1 atom of N2,N2-dimethylguanine, with a second water-mediated hydrogen bond between the N-3 atom of dideoxycytosine and the O-6 atom of N2,N2-dimethylguanine. A crystal structure of Dpo4 with dTTP opposite template N2,N2-dimethylguanine reveals a wobble orientation
ternary enzyme/DNA/dNTP complexes
ternary polymerase-DNA-dNTP, dGTP and/or dATP, complexes for three template-primer DNA sequences, 18-mer-primer 13-mer sequences containing 1,N2-propanodeoxyguanosine, with bound Ca2+, X-ray diffraction structure determination and analysis at 2.4-2.7 A resolution, modelling
the enzyme is mixed with DNA (1:1.2 molar ratio) in 20 mM Tris-HCl buffer (pH 8.0, 25 °C) containing 60 mM NaCl, 4% glycerol (v/v), and 5 mM 2-mercaptoethanol and then placed on ice for 1 h prior to incubation with 5 mM MgCl2 and 1 mM dGTP. Crystals are grown using the sitting drop/vapor-diffusion method with the reservoir solution containing 20 mM Tris-HCl (pH 8.0 at 25 °C), 15% polyethylene glycol 3350 (w/v), 60 mM NaCl, 5 mM MgCl2, and 4% glycerol (v/v). Two crystal structures of Dpo4 with a template N2-NaphG (in a post-insertion register opposite a 3-terminal C in the primer) are solved. One shows N2-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 “little finger” domain. The Hoogsteen face is within hydrogen bonding distance of the N4 atoms of the cytosine opposite N2-NaphG and the cytosine at the 2 position. The second structure shows N2-NaphG in an anti conformation with the primer terminus largely disordered
the structure of the enzyme bound to G*T-mispaired primer template in the presence of an incoming nucleotide is solved. As a control the structure of the enzyme bound to a matched A-T base pair at the primer terminus is also determined. The structures offer a basis for the low efficiency of the enzyme in extending a G*T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide
three crystal structures of a the enzyme in complex with Pt-GG DNA (1,2-intrastrand covalent linkage, cis-Pt-1,2-d(GpG)) at 2.9, 1.9, and 2.0 A resolution, respectively. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3'G (first insertion) and 5'G (second insertion) of Pt-GG, and the primer extension beyond the lesion site
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D424A
mutation in polymerase active site, the polymerase activity is reduced more than 30fold compared to the wild-type activity
D424A/D542A
mutation in polymerase active site, complete inactivation of polymerase activity
D542A
mutation in polymerase active site, the polymerase activity is reduced more than 50fold compared to the wild-type activity
N188W
kcat/Km for dCTP is 2.9fold compared to kcat/Km of wild-type enzyme
N249Y
exhibits increased catalytic activity when compared to the wild-type enzyme, the mutation decreases the affinity for NAD(H) cofactor
R322H
the His332 mutant exhibits faster forward rate constants relative to wild-type Dpo4. The kpol values for the His332 mutant incorporation opposite G and 8-oxoG are 3.6- and 4.6fold faster than for wild-type Dpo4. The nucleotide binding affinity trend is opposite that of wild-type Dpo4, Glu332, and Leu332, with tighter dCTP binding during bypass of G. As in the case of Ala332, the kinetic analysis indicates that His332 inserts dCTP opposite G with slightly greater efficiency than opposite 8-oxoG
R322L
kpol (forward rate of polymerization) values for the Leu332 mutant incorporation opposite G and 8-oxoG are 4.1- and 1.9fold higher than those for wild-type Dpo4. The Leu332 mutant is about 2fold more efficient at incorporation of dCTP opposite 8-oxoG compared with G
R331A/R322A
mutant has lower forward rate constants relative to wild-type enzyme for both G and 8-oxoG. The double mutant-catalyzed insertion of dCTP opposite 8-oxoG is about 4fold higher than dCTP insertion opposite G. The measured binding affinity of dCTP is tighter than that of wild-type Dpo4 for unmodified DNA, but the binding affinity of dCTP opposite 8-oxoG is similar to that observed for wild-type enzyme. The catalytic efficiency for dCTP incorporation increases about 4fold for unmodified DNA and decreases about 2fold for 8-oxoG-modified DNA. The mutant fails to incorporate dATP opposite 8-oxoG in the presteady-state experiments. It inserts dCTP opposite 8-oxoG with an about 200fold greater efficiency than it does dATP and the steady-state efficiency of dATP incorporation is decreased about 27fold relative to the wild-type enzyme
R332A
mutant enzyme displays a higher affinity (lower KD,dCTP) for dCTP when bound to the unmodified DNA compared with the KD,dCTP measured for mutant-catalyzed incorporation of dCTP opposite 8-oxoG. Wild-type enzyme shows a greater affinity for dCTP opposite to 8-oxoG-modified DNA. The catalytic efficiency of the mutant is 23fold higher at incorporation of C opposite G and 1.2fold lower than wild-type enzyme in dCTP incorporation opposite 8-oxoG
R332E
mutant enzyme retains fidelity against bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) that is similar to wild enzyme. A crystal structure of the mutant and 8-oxoG:C pair reveals water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The kpol (forward rate of polymerization) value for dCTP incorporation opposite G is 4.8fold higher than for wild-type enzyme. The kpol (forward rates of polymerization) value for dCTP incorporation opposite 8-oxoG is 3.5fold higher than wild-type enzyme insertion of dCTP opposite 8-oxoG. The catalytic efficiency of the Glu332 mutant is 2.3fold greater than wild-type Dpo4 for dCTP incorporation opposite G but 1.7fold less efficient than wild-type Dpo4 for incorporation opposite 8-oxoG
T239W
additional information
generaion of chimeras of Sulfolobus solfataricus DNA polymerase Dpo4 and Sulfolobus acidocaldarius DNA polymerase Dbh in which their little finger domains have been interchanged. Interestingly, by replacing the little finger domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. The unique but variable little finger domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member