Information on EC 2.7.7.31 - DNA nucleotidylexotransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.31
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RECOMMENDED NAME
GeneOntology No.
DNA nucleotidylexotransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
deoxynucleoside triphosphate + DNAn = diphosphate + DNAn+1
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
-
-
-
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polymerization
SYSTEMATIC NAME
IUBMB Comments
nucleoside-triphosphate:DNA deoxynucleotidylexotransferase
Catalyses template-independent extension of the 3'- end of a DNA strand by one nucleotide at a time. Cannot initiate a chain de novo. Nucleoside may be ribo- or 2'-deoxyribo-.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-67-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
rainbow trout
-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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enzyme is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. Enzyme is ubiquitylated by the Cul3-based ubiquitylation system in vitro and may also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Ubiquitylation inhibits the nucleotidyltransferase activity
physiological function
in Chinese hamster ovary cells, ectopic expression of the enzmye efficiently promotes N-additions at the junction of chromosomal double-strand breaks generated by the meganuclease I-SceI. The size of the N-additions is comparable with that at V(D)J junctions. No N-addition is observed in KU80- or XRCC4-deficient cells
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-[2-N-(9-fluorenylmethoxycarbonyl)aminoethylphosphonyl]-beta,gamma-difluoromethylenediphosphonate + DNAn
diphosphate + ?
show the reaction diagram
-
synthetic substrate
-
-
?
alpha-[2-N-(9-fluorenylmethoxycarbonyl)aminoethylphosphonyl]-beta,gamma-diphosphate + DNAn
diphosphate + ?
show the reaction diagram
-
synthetic substrate
-
-
?
D-beta-deoxythymidine-triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
-
-
-
?
dATP + DNAn
diphosphate + DNAn+1
show the reaction diagram
terminal transferase activity at the 3'-OH extremity of the nicking site
-
-
?
deoxynucleoside triphosphate + DNA
diphosphate + DNAn+1
show the reaction diagram
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
dTTP + DNAn
?
show the reaction diagram
-
-
-
-
?
d[5-(3-aminophenyl)]CTP + DNA
diphosphate + ?
show the reaction diagram
5-substituted dCTPs give products elongated by 1 to 5 nucleotides, with most intense bands corresponding to 2-3 extra nucleotides
-
-
?
d[5-(3-aminophenyl)]UTP + DNA
diphosphate + ?
show the reaction diagram
5-substituted dUTPs give products elongated by 4 to 5 nucleotides, the maximum length of the tail is about 14 nucleotides
-
-
?
d[5-(3-nitrophenyl)]CTP + DNA
diphosphate + ?
show the reaction diagram
5-substituted dCTPs give products elongated by 1 to 5 nucleotides, with most intense bands corresponding to 2-3 extra nucleotides
-
-
?
d[5-(3-nitrophenyl)]UTP + DNA
diphosphate + ?
show the reaction diagram
5-substituted dUTPs give products elongated by 4 to 5 nucleotides, the maximum length of the tail is about 14 nucleotides
-
-
?
d[7-deaza-7-(3-aminophenyl)]ATP + DNA
diphosphate + ?
show the reaction diagram
the most intense band observed for reaction with 7-deaza,7-substituted dATPs corresponds to one nucleotide attached, but the products are rather polydisperse in lengths, reaching several tens of extra nucleotides
-
-
?
d[7-deaza-7-(3-aminophenyl)]GTP + DNA
diphosphate + ?
show the reaction diagram
-
-
-
?
d[7-deaza-7-(3-nitrophenyl)]ATP + DNA
diphosphate + ?
show the reaction diagram
the most intense band observed for reaction with 7-deaza,7-substituted dATPs corresponds to one nucleotide attached, but the products are rather polydisperse in lengths, reaching several tens of extra nucleotides
-
-
?
d[7-deaza-7-(3-nitrophenyl)]GTP + DNA
diphosphate + ?
show the reaction diagram
most useful substrate, efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide
-
-
?
ribonucleoside triphosphate + DNAn
diphosphate + ?
show the reaction diagram
-
-
-
-
?
ssDNA + ferrocene-labeled dideoxy-UTP
ssDNA-ddUFc + 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
deoxynucleoside triphosphate + DNA
diphosphate + DNAn+1
show the reaction diagram
deoxynucleoside triphosphate + DNAn
diphosphate + DNAn+1
show the reaction diagram
-
the long murine terminal deoxynucleotidyltransferase isoform does not play a role in the control of V(D)J junctional diversity
-
-
?
ssDNA + ferrocene-labeled dideoxy-UTP
ssDNA-ddUFc + diphosphate
show the reaction diagram
-
-
-
-
?
additional information
?
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alternative splicing seems to regulate TdT isoform expression. Both long isoforms, hTdTL1 and hTdTL2 have 3'-5' exonuclease activity. Overexpression of the short isoform hTdTS or the long isoform hTdTL2 greatly reduces the efficiency of recombinantion, which is reverted to normal levels by simultaneous expression of both enzymes
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R,2S)-1-(6-aminopurin-9-yl)-2-(Hydroxymethyl)cyclopentane
-
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(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate
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-
1,10-phenanthroline
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not m-isomer
2',3'-dideoxyadenosine
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inhibition is stronger with (dA)12-18 as primer than with heat denatured DNA, substrate ATP, 50% inhibition at 0.006 mM
2',3'-dideoxycytidine
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inhibition is stronger with (dA)12-18 as primer than with heat denatured DNA, substrate CTP, 50% inhibition at 0.002 mM
2',3'-dideoxyguanosine
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inhibition is stronger with (dA)12-18 as primer than with heat denatured DNA, substrate GTP, 50% inhibition at 0.005 mM
2',3'-dideoxythymidine
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inhibition is stronger with (dA)12-18 as primer than with heat denatured DNA, substrate TTP, 50% inhibition at 0.020 mM
2',3'-dideoxythymidine 5'-triphosphate
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2,2'-dipyridyl
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-
3'-azido-TTP
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3'-dATP
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selective inhibitor, inactive in DNA-synthesizing systems both with DNA polymerase alpha or beta
3'-Deoxy-3'-fluorothymidine 5'-triphosphate
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5-(hydroxymethyl)-2-furfural
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HMF, a pyrolysate of carbohydrate isolated from instant coffee (CoVea arabica L.)
6-(1-phenyl-1H-indol-3-yl)-2,4-dioxy-5-hexenoic acid
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IC50: 0.0472 mM
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6-(1-phenylmethyl-1H-indol-3-yl)-2,4-dioxy-5-hexenoic acid
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IC50: 0.0079 mM
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6-(4-benzoyl-1-phenylmethyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acid
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IC50: 0.0162 mM
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9-alpha-Arabinofuranosyladenine 5'-triphosphate
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9-beta-Arabinofuranosyladenine 5'-triphosphate
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-
Ap5A
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Baicalin
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competitive versus the DNA primer and non-competitive versus the dNTP substrate, IC50: 0.0186 mM
cordycepin
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selectively cytotoxic against TdT+ leukemic cells, particularly in combination with the adenosine deaminase inhibitor deoxycoformycin
cysteine
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D-/L-carbocyclic dideoxynucleoside triphosphates
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dATP
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mechanism of Mn2+-dependent inhibition
ddATP
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ddCTP
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ddGTP
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ddTTP
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diadenosine 5',5'-diphosphate
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diadenosine 5',5'-hexaphosphate
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diadenosine 5',5'-pentaphosphate
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diadenosine 5',5'-tetraphosphate
diadenosine 5',5'-triphosphate
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diallyl trisulfide
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diphosphate
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EDTA
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genistein
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competitive versus the DNA primer and non-competitive versus the dNTP substrate, IC50: 0.0287 mM
High ionic strength
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histidine
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L-beta-deoxythymidine-triphosphate
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i.e. L-dTTP, competitive
L-dTTP
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Mercaptoacetate
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Metal chelators
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N-ethylmaleimide
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NH4+
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Oligo(dA)
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product inhibition
P1,P5-diadenosine 5'-pentaphosphate
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-
phosphate
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pyran
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Streptolydigin
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does not significantly inhibit DNA polymerase alpha, beta, and gamma, or RNA polymerase, non-competitive to (dA)12-18 and dGTP
TdT interacting factor 1
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negatively regulates TdT activity, TdT interacting factor 1 binds to the Pol beta-like region in TdT and blocks TdT access to DNA ends
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triethylenetetramine
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-
additional information
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Allium sativa extract is a mixture of diallyl trisulfide, diallyl tetrasulfide, and diallyl pentasulfide exhibiting an IC50 value of 0.371 mM at 37C and a KI value of 0.0121 mM at 37C
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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stimulates
sulfhydryl compound
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required
TdT interacting factor 1
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releases TdT in the presence of dsDNA to exhibit TdT activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000009
alpha-[2-N-(9-fluorenylmethoxycarbonyl)aminoethylphosphonyl]-beta,gamma-difluoromethylenediphosphonate
-
37C, pH 7.2
0.000033
alpha-[2-N-(9-fluorenylmethoxycarbonyl)aminoethylphosphonyl]-beta,gamma-diphosphate
-
37C, pH 7.2
0.1
D-beta-deoxythymidine-triphosphate
-
pH 6.6, 37C, primer oligo(dT)20
0.004 - 0.01
dATP
0.071
dCTP
-
37C, primer (dA)12-18
0.083 - 0.114
dGTP
0.00008 - 0.0002
DNA
0.000032 - 1.02
dTTP
1
homopolymer primers
-
-
-
0.02
oligo(dA)12-18
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37C, pH 8.3
0.001
oligonucleotide primers
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-
-
0.0003 - 0.0025
poly(dA)50
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.833
dATP
Bos taurus
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-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0005
(1R,2S)-1-(6-aminopurin-9-yl)-2-(Hydroxymethyl)cyclopentane
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-
0.005
(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate
-
-
0.01
3'-azido-TTP
-
-
0.00025
Ap5A
-
-
0.00981 - 0.0152
Baicalin
0.0009 - 0.006
cordycepin
-
-
0.005 - 0.01
D-/L-carbocyclic dideoxynucleoside triphosphates
-
-
-
0.0013
ddATP
-
-
0.0028
ddCTP
-
-
0.0013
ddGTP
-
-
0.0175
ddTTP
-
-
0.0231 - 0.0338
genistein
0.06
L-dTTP
-
pH 6.6, 37C, primer oligo(dT)20
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0055
5-(hydroxymethyl)-2-furfural
Bos taurus
-
-
0.0472
6-(1-phenyl-1H-indol-3-yl)-2,4-dioxy-5-hexenoic acid
Bos taurus
-
IC50: 0.0472 mM
-
0.0079
6-(1-phenylmethyl-1H-indol-3-yl)-2,4-dioxy-5-hexenoic acid
Bos taurus
-
IC50: 0.0079 mM
-
0.0162
6-(4-benzoyl-1-phenylmethyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acid
Bos taurus
-
IC50: 0.0162 mM
-
0.0186
Baicalin
Homo sapiens
-
competitive versus the DNA primer and non-competitive versus the dNTP substrate, IC50: 0.0186 mM
0.144
diallyl trisulfide
Bos taurus
-
at 37C
0.0287
genistein
Homo sapiens
-
competitive versus the DNA primer and non-competitive versus the dNTP substrate, IC50: 0.0287 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
assay at
37
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assay at
75
the nucleotidyl-transferase activity of Rep75 is less thermophilic than its nicking activity (105C)
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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exclusively express hTdTS and hTdTl2
Manually annotated by BRENDA team
and the tissue around the intestine; intestine and the tissue around the intestine
Manually annotated by BRENDA team
pronephros and mesonephros
Manually annotated by BRENDA team
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transformed, hTdTL2 expression is restricted to transformed lymphoid cell lines
Manually annotated by BRENDA team
the primary lymphoid organs
Manually annotated by BRENDA team
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exclusively express hTdTS and hTdTl2
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32360
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equilibrium sedimentation
40000 - 45000
-
-
42000 - 60000
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gel filtration, the 2 subunits of the calf thymus enzyme reported earlier may be proteolytic products derived from a single polypeptide of MW 60000, which may be the native form
44000
-
2 high MW forms: 58000 and 45000 and one two subunit form of 44000 MW
57000
-
2 MW forms: 45000 and 57000, gel filtration
58000
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2 high MW forms: 58000 and 45000 and one two subunit form of 44000 MW
62000
-
gel filtration
79000
-
-
additional information
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 57000, deduced from gene sequence
dimer
-
1 * 8000 (alpha) + 1 * 26500 (beta), SDS-PAGE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
-
stable at
643200, 643201
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
60 min, 60% residual activity of short isoform, 20% residual activity of long isoform
40
-
not stable above
100
1 h, no loss of activity after 1 h
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
not stable in urea, SDS and organic solvents
-
rapid loss of activity can be eliminated by addition of albumin to the reaction mixture
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
not stable in organic solvents
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 40% loss of activity after 7 months
-
-20C, 50 mM potassium phosphate buffer, pH 7.0, stable for 9 years
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 high MW forms: 58000 and 45000 and one two subunit form of 44000 MW; calf
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2 MW forms: 45000 and 57000
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calf; single step immunoaffinity purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli TOP10 F' cells
expression in Escherichia coli
-
expression in Escherichia coli and in 293T cell
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
del1-232
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mutant shows no appreciable activity
del1-280
-
mutant shows no appreciable activity
del421-501
-
mutant shows no appreciable activity
del453-501
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mutant shows no appreciable activity
G257A/V258A/G259A
-
mutant shows no appreciable activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
medicine