Information on EC 2.7.7.B16 - DNA primase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.B16
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
DNA primase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
dNTP + n dNTP = dN(pdN)n + n diphosphate
show the reaction diagram
NTP + n NTP = N(pN)n + n diphosphate
show the reaction diagram
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Q9V292: small subunit, Q9V291: large subunit
Q9V292 and Q9V291
SwissProt
Manually annotated by BRENDA team
Q9P9H1: small subunit, Q8U4H7: large subunit
Q9P9H1 and Q8U4H7
SwissProt
Manually annotated by BRENDA team
O57934: small subunit, O57935: large subunit
O57934 and O57935
SwissProt
Manually annotated by BRENDA team
Q5JJ72: small subunit and Q5JJ73: large subunit
Q5JJ72 and Q5JJ73
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + ATP
A(pA)n + n diphosphate
show the reaction diagram
Q5JJ72 and Q5JJ73
oligo(dT)30 supports extensive DNA and RNA synthesis. Oligo(dT)30 supports the synthesis of shorter RNA chains than those formed in the presence of oligo(dC)30 as well as the production of higher levels of RNA than DNA
-
-
?
ATP + n ATP
A(pA)n + n diphosphate
show the reaction diagram
dATP + dATP
dA(pdA)n + n diphosphate
show the reaction diagram
Q5JJ72 and Q5JJ73
oligo(dT)30 supports extensive DNA and RNA synthesis
-
-
?
dATP + glycerol
dAMP-glycerol + diphosphate
show the reaction diagram
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the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dATP + Tris
dAMP-Tris + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dCTP + glycerol
dAMP-glycerol + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dCTP + Tris
dAMP-Tris + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dGTP + glycerol
dGMP-glycerol + diphosphate
show the reaction diagram
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the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dGTP + n dGTP
dG(pdG)n + n diphosphate
show the reaction diagram
dGTP + Tris
dGMP-Tris + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dNTP + n dNTP
dN(pdN)n + n diphosphate
show the reaction diagram
dNTP + n NTP
(dNTP)n+1 + n diphosphate
show the reaction diagram
Q9V292 and Q9V291
DNA primase has comparable affinities for ribonucleotides and deoxyribonucleotides. The Pabp41 subunit alone has no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increases the rate of DNA synthesis but decreases the length of the DNA fragments synthesized and confers RNA synthesis capability. DNA primase also displayed DNA polymerase, gapfilling, and strand-displacement activities
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-
?
dTTP + glycerol
dTMP-glycerol + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
dTTP + Tris
dTMP-Tris + diphosphate
show the reaction diagram
-
the products are formed by the p41 catalytic subunit alone and the p41-p46 complex in the absence of a DNA template. They are not formed with preparations containing the catalytically inactive p41 subunit
-
-
?
GTP + n GTP
G(pG)n + n diphosphate
show the reaction diagram
NTP + n NTP
N(pN)n + n diphosphate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
dNTP + n dNTP
dN(pdN)n + n diphosphate
show the reaction diagram
NTP + n NTP
N(pN)n + n diphosphate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
required
K+
activity requires divalent cations such Mg2+, Mn2+ or Zn2+, and is additionally stimulated by the monovalent cation K+
additional information
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Rep245 polymerase activity is strictly dependent on divalent cations (Mg2+ or Mn2+), Zn2+ cations do not support the DNA polymerization activity of Rep245
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dATP
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synthesis of the short RNA chains is inhibited at all levels of dATP added, and the size of oligo(rA) chains formed and the amount of ATP incorporated are reduced
dGTP
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dGTP at a molar ratio of dGTP to dATP or rATP of 10:1 inhibits both DNA and RNA synthesis. Lower molar ratios of dGTP:rATP (0.1:1) inhibit ATP incorporation by 91%, whereas dATP incorporation is reduced by 8%
GTP
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dGTP at a molar ratio of dGTP to dATP or rATP of 10:1 reduces dATP incorporation by 43% and ATP incorporation by 92%
Mn2+
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RNA synthesis with the Thermococcus kodakaraensis primase complex is stimulated about 2fold by the presence of Mn2+, whereas the size of RNA chains is marginally affected. DNA synthesis is slightly inhibited by Mn2+
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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in the presence of high levels of ATP (ATP:dATP molar ratio of 10:1), dAMP incorporation is stimulated 3-fold, although the size of dAMP-labeled products formed is reduced
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08 - 0.09
ATP
0.03 - 0.04
dATP
0.05
dGTP
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pH 8.0, 60°C, in presence of oligo(dC)
0.028
dNTP
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pH 8.0, 60°C, in presence of M13 ssDNA as template
0.25
GTP
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pH 8.0, 60°C, in presence of oligo(dC)
0.0032 - 0.085
NTP
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00055 - 0.022
NTP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000013 - 0.0068
NTP
1383
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
Q9UWW1 AND Q97Z83 AND Q97ZS7
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
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pH 7.0: about 40% of maximal activity, pH 8.5: about 50% of maximal activity
7.5 - 8.5
pH 7.5: about 40% of maximal activity, pH 8.5: about 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
Q9UWW1 AND Q97Z83 AND Q97ZS7
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80
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template-free synthesis activity
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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coupled assay with DNA polymerase B
45 - 75
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45°C: about 40% of maximal activity, 75°C: about 40% of maximal activity
50 - 90
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50°C: about 45% of maximal activity, 90°C: about 35% of maximal activity
55 - 70
Q9UWW1 and Q97Z83
polymerization reactions on C35 or C34ddC with rGTP. Both the size and the quantity of the products on C35 increase, while synthesis on C34ddC drastically decreases, when the reaction temperature is raised from 55°C to 70°C
68 - 80
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activity range of template?free synthesis, which is highly temperature-dependent
additional information
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delayed kinetics of the reaction at low temperature
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
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x * 29000, Rep245 domain containing the N-terminal domain of the pIT3 replication protein encompassing residues 31–245, SDS-PAGE
36000
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1 * 36000 + 1 * 38000, SDS-PAGE
38000
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1 * 36000 + 1 * 38000, SDS-PAGE
40772
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1 * 46209 (large subunit Pfup46) + 1 * 40772 (small subunit Pfup41), calculated from sequence
41000
Q9V292 and Q9V291
1 * 41000 + 1 * 46000, calculated from sequence
41800
x * 41800, calculated from sequence
46000
Q9V292 and Q9V291
1 * 41000 + 1 * 46000, calculated from sequence
46209
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1 * 46209 (large subunit Pfup46) + 1 * 40772 (small subunit Pfup41), calculated from sequence
80000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
heterotrimer
Q9UWW1 AND Q97Z83 AND Q97ZS7
archaea encode a eukaryotic-type primase comprising a catalytic subunit, PriS, and a noncatalytic subunit, PriL and PriX. PriX is a diverged homologue of the C-terminal domain of PriL but lacks the iron-sulfur cluster. PriX, PriL and PriS form a stable heterotrimer (PriSLX). Both PriSX and PriSLX show far greater affinity for nucleotide substrates and are substantially more active in primer synthesis than the PriSL heterodimer. PriL, but not PriX, facilitates primer extension by PriS. The catalytic activity of PriS is modulated through concerted interactions with the two noncatalytic subunits in primer synthesis. PriX subunit residues 26-54 are in a flexible region. PriX residues 55-154 fold into a single domain containing 6 helices, which form a compact core stabilized by extensive hydrophobic interactions. The overall structure of the PriX protein is quite unique, sequence and three-dimensional structure comparisons, overview
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
vapor diffusion in sitting drops at 20°C. Crystal structure of the catalytic primase subunit, 2.3 A resolution
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cocrystalization with uridine 5'-triphosphate allowing confirmation of the location of the active site. Construction of a model between the DNA primase and a primer/template DNA based on the complex structure for the primer synthesis
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crystallographic studies of of the N-terminal domain (NTD) of PriL (PriLNTD; residues 1–222) that bind to PriS, 2.9 A resolution
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hanging-drop vapor diffusion method at 20°C, with polyethylene glycol 8000 as the precipitant. The crystals belong to the P3(2)21 with unit-cell parameters a = b = 77.8, c = 129.6 A, and alpha = beta = 90°, gamma = 120°. Crystals of the selenomethionine derivative are obtained by means of a cross-seeding method using native crystals. The data for the native and selenomethionine-substituted crystals are collected to 1.8 and 2.2 A resolution
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structure-function analysis of the pRN1 primase-polymerase domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues
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hanging drop vapor diffusion at 18°C, the structure provides the first three-dimensional description of the large subunit and its interaction with the small subunit
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purified recombinant PriX deletion mutant 26-154, X-ray diffraction strutcure determination and analysis at 1.95 A resolution, crystallization of the full-length PriX is unsuccessful
Q9UWW1 AND Q97Z83 AND Q97ZS7
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
1 h, enzyme is stable
70
10 min, 20% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable to repeated freezing and thawing
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified following overexpression in Escherichia coli
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recombinant His-tagged DnaG from Escherichia coli by nickel affinity chromatography, co-purification of Strep-tagged Csl4 and His-tagged Rrp4, Rrp41, Rrp42 and DnaG
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recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli, copurification of the His6-tagged wild-type enzyme with His6-tagged Rrp4, Csl4, Rrp41 and Rrp42, and Strep-tagged Csl4
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the gene for the p58-like protein (gene product: Pfup46), expression in Escherichia coli
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expression in Escherichia coli
gene dnaG, sequence comparisons, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli, coexpression with His6-tagged Rrp4, Csl4, Rrp41 and Rrp42, and Strep-tagged Csl4
genes priL, priS, and SSO0502, DNA and amino acid sequence determination and analysis of PriX, phylogenetic analysis and tree, recombinant expression of a deletion mutant PriX protein containing amino acid residues 26-154
Q9UWW1 AND Q97Z83 AND Q97ZS7
hexa-histidine tagged enzyme is expressed in Escherichia coli
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overexpression in Escherichia coli
overexpression in Escherichia coli as a fusion protein with a hexa-histidine tag
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purified following overexpression in Escherichia coli
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recombinant expression of His-tagged DnaG in Escherichia coli
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small and large subunit are co-expressed in Escherichia coli as hexa-Histidine tagged proteins
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y155A/Y156A/I157A
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mutation reduces PriS binding 1000fold
D101A/D103A
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the catalyric site mutant enzyme does not exhibit any detectable catalytic activity, even when higher concentrations of enzyme are utilised. The result demonstrate that the terminal transferase-like activity of PriSL is dependent on the catalytic activity of the primase. D101 and D103 are necessary for PriSL catalytic activity
F164E(PriS)
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mutantion in small subunitPriS shows considerably weakened subunit association
F164G/I199K(PriS)_F142E/L163E(PriL)
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double mutations of F164G/I199K in small subunit PriS and F142E/L163E in large subunit PriL abolishes the PriS-PriL interaction
G165I(PriS)
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mutantion in small subunitPriS shows partial destabilization of the complex
K6A/Y7A
site-directed mutagenesis of the N-terminal domain
N175A/R176
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the affinity of DNA-binding site mutant for NTPs is approximately tenfold lower than that of the wild-type primase and that its enzymatic capability is diminished. Therefore, the mutation of N175 and R176 does not alter the DNA binding properties of the primase but modifies its affinity for free NTPs
R84A/R85A(PriL)
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mutation in large subunit shows a marked reduction in the size and amount of RNA product synthesized
D101A/D103A
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the catalyric site mutant enzyme does not exhibit any detectable catalytic activity, even when higher concentrations of enzyme are utilised. The result demonstrate that the terminal transferase-like activity of PriSL is dependent on the catalytic activity of the primase. D101 and D103 are necessary for PriSL catalytic activity
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F164E(PriS)
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mutantion in small subunitPriS shows considerably weakened subunit association
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F164G/I199K(PriS)_F142E/L163E(PriL)
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double mutations of F164G/I199K in small subunit PriS and F142E/L163E in large subunit PriL abolishes the PriS-PriL interaction
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G165I(PriS)
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mutantion in small subunitPriS shows partial destabilization of the complex
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N175A/R176
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the affinity of DNA-binding site mutant for NTPs is approximately tenfold lower than that of the wild-type primase and that its enzymatic capability is diminished. Therefore, the mutation of N175 and R176 does not alter the DNA binding properties of the primase but modifies its affinity for free NTPs
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R84A/R85A(PriL)
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mutation in large subunit shows a marked reduction in the size and amount of RNA product synthesized
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additional information