Information on EC 2.7.7.B16 - DNA primase

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The expected taxonomic range for this enzyme is: Archaea, Bacteria

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 subuntit, 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
physiological function
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
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-
?
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
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-
?
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
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-
?
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
Q5JJ72 and Q5JJ73
oligo(dC)30 supports extensive DNA and RNA synthesis. Of the four homo-oligodeoxynucleotides, 30 nt in length oligo(dC)30 is the most effective template supporting extensive DNA synthesis with dGTP. dGTP incorporation exceeds the level of oligo(dC)30 template added, and the lengths of DNA chains are 100 nt
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-
?
dGTP + Tris
dGMP-Tris + 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
-
-
?
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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interaction of Sulfolobus solfataricus DnaG primase (SsoDnaG) with the replicative minichromosome maintenance helicase (SsoMCM) on DNA. The site of interaction is mapped. The complex of SsoDnaG with SsoMCM stimulates the ATPase activity of SsoMCM but leaves the priming activity of SsoDnaG unchanged
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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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synthetic function is specifically activated by thymine-containing synthetic bubble structures that mimic early replication intermediates
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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, 60C, in presence of oligo(dC)
0.028
dNTP
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pH 8.0, 60C, in presence of M13 ssDNA as template
0.25
GTP
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pH 8.0, 60C, in presence of oligo(dC)
0.025 - 0.085
NTP
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00055
NTP
Sulfolobus solfataricus
P95980
KM-value is determined by measuring the priming rate at different concentrations of NTPs, 70C, pH 8.5, in presence of M13 ssDNA as template, wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000033 - 0.0063
NTP
1383
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 75
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45C: about 40% of maximal activity, 75C: about 40% of maximal activity
50 - 90
50C: about 45% of maximal activity, 90C: 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 55C to 70C
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
additional information
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the Pfup46 protein increases the affinity of the primase to DNA by forming a complex with the catalytic Pfup41 subunit
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
vapor diffusion in sitting drops at 20C. 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 1222) that bind to PriS, 2.9 A resolution
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hanging-drop vapor diffusion method at 20C, 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 18C, the structure provides the first three-dimensional description of the large subunit and its interaction with the small subunit
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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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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
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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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
E175Q
mutation drastically reduces priming activity, low level of DNA binding 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
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