Information on EC 3.2.2.29 - thymine-DNA glycosylase

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

EC NUMBER
COMMENTARY hide
3.2.2.29
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RECOMMENDED NAME
GeneOntology No.
thymine-DNA glycosylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolyses mismatched double-stranded DNA and polynucleotides, releasing free thymine.
show the reaction diagram
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-
-
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SYSTEMATIC NAME
IUBMB Comments
thymine-DNA deoxyribohydrolase (thymine-releasing)
Thymine-DNA glycosylase is part of the DNA-repair machinery. Thymine removal is fastest when it is from a G/T mismatch with a 5'-flanking C/G pair. The glycosylase removes uracil from G/U, C/U, and T/U base pairs faster than it removes thymine from G/T [3].
CAS REGISTRY NUMBER
COMMENTARY hide
149565-68-4
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
additional information
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opposing roles of CBP/p300 and PKCalpha in regulating the DNA repair functions of TDG, the interplay of acetylation and phosphorylation of TDG in vivo may be critically important in the maintenance of CpG dinucleotides and epigenetic regulation
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
show the reaction diagram
5-bromocytosine-mismatched double-stranded DNA + H2O
5-bromouracil + double-stranded DNA with abasic site
show the reaction diagram
-
hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site
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-
?
5-bromouracil-mismatched double-stranded DNA + H2O
5-bromouracil + double-stranded DNA with abasic site
show the reaction diagram
5-carboxylcytosine mismatched double-stranded DNA + H2O
5-carboxylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-carboxylcytosine-mismatched double-stranded DNA + H2O
5-carboxylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-chlorouracil-mismatched double-stranded DNA + H2O
5-chlorouracil + double-stranded DNA with abasic site
show the reaction diagram
5-fluorocytosine-mismatched double-stranded DNA + H2O
5-fluorocytosine + double-stranded DNA with abasic site
show the reaction diagram
-
hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site
-
-
?
5-fluorouracil-mismatched double-stranded DNA + H2O
5-fluorouracil + double-stranded DNA with abasic site
show the reaction diagram
5-formylcytosine-mismatched double-stranded DNA + H2O
5-formylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-hydroxcytosine-mismatched double-stranded DNA + H2O
5-hydroxycytosine + double-stranded DNA with abasic site
show the reaction diagram
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hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site
-
-
?
5-hydroxymethyluracil-mismatched double-stranded DNA + H2O
5-hydroxymethyluracil + double-stranded DNA with abasic site
show the reaction diagram
5-hydroxyuracil-mismatched double-stranded DNA + H2O
5-hydroxyuracil + double-stranded DNA with abasic site
show the reaction diagram
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removes 5-hydroxyuracil from G/5-hydroxyuracil mismatches
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-
?
5-methylcytosine-mismatched double-stranded DNA + H2O
5-methylcytosine + double-stranded DNA with abasic site
show the reaction diagram
8-(hydroxymethyl)-3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
8-(hydroxymethyl)-3,N4-ethenocytosine + double-stranded DNA with abasic site
show the reaction diagram
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TDG is able to excise the 8-(hydroxymethyl)-3,N4-ethenocytosine from DNA. TDG activity displays a marked preference of guanine opposite to 8-(hydroxymethyl)-3,N4-ethenocytosine over any other bases. TDG does not show any detectable activity toward 3,N4-ethanocytosine when placed in various neighboring sequences, including the 5'-CpG site
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-
?
cytosine-mismatched double-stranded DNA + H2O
cytosine + double-stranded DNA with abasic site
show the reaction diagram
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paired with guanine
-
-
?
double-stranded DNA + H2O
?
show the reaction diagram
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thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine. This suggests a main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites
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-
?
thymine glycol -mismatched double-stranded DNA + H2O
thymine glycol + double-stranded DNA with abasic site
show the reaction diagram
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thymine glycol from G/thymine glycol mismatches
-
-
?
thymine glycol-mismatched double-stranded DNA + H2O
thymine glycol + double-stranded DNA with abasic site
show the reaction diagram
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oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. TDG and methyl-CpG-binding protein 4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides
-
-
?
thymine-mismatched double-stranded DNA + H2O
thymine + double-stranded DNA with abasic site
show the reaction diagram
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
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
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
show the reaction diagram
-
3,N4-ethenocytosine is recognized and efficiently excised by hTDG. The enzyme may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability
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-
?
5-bromouracil-mismatched double-stranded DNA + H2O
5-bromouracil + double-stranded DNA with abasic site
show the reaction diagram
-
potential role played by human TDG in the cytotoxic effects of 5-chlorouracil and 5-bromouracil incorporation into DNA, which can occur under inflammatory conditions
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-
?
5-carboxylcytosine mismatched double-stranded DNA + H2O
5-carboxylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-carboxylcytosine-mismatched double-stranded DNA + H2O
5-carboxylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-chlorouracil-mismatched double-stranded DNA + H2O
5-chlorouracil + double-stranded DNA with abasic site
show the reaction diagram
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potential role played by human TDG in the cytotoxic effects of 5-chlorouracil and 5-bromouracil incorporation into DNA, which can occur under inflammatory conditions
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-
?
5-formylcytosine-mismatched double-stranded DNA + H2O
5-formylcytosine + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
5-hydroxymethyluracil-mismatched double-stranded DNA + H2O
5-hydroxymethyluracil + double-stranded DNA with abasic site
show the reaction diagram
-
-
-
-
?
double-stranded DNA + H2O
?
show the reaction diagram
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thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine. This suggests a main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites
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-
?
thymine glycol-mismatched double-stranded DNA + H2O
thymine glycol + double-stranded DNA with abasic site
show the reaction diagram
-
oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. TDG and methyl-CpG-binding protein 4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides
-
-
?
thymine-mismatched double-stranded DNA + H2O
thymine + double-stranded DNA with abasic site
show the reaction diagram
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4Fe-4S cluster
the enzyme contains a binding motif for the 4Fe-4S cluster
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
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slight inhibition of the enzymic activity
Co2+
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complete inhibition at 5 mM
Cu2+
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complete inhibition at 5 mM
Mg2+
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slight inhibition of the enzymic activity
Mn2+
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complete inhibition at 5 mM
NaCl
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NaCl completely inhibits the thymine removal at 0.4 M
Ni2+
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complete inhibition at 5 mM
Zn2+
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complete inhibition at 5 mM
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
SUMO-1
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TDG interacts with, but also is modified by SUMO-1 and SUMO-3, SUMOs are small ubiquitin like modifiers, small polypeptides structurally related to ubiquitin that interact with and/or are attached to other proteins. SUMO conjugation involves Lys330 located in a C-terminal SUMOylation consensus motif, VKEE, it is ATP-dependent and, when performed in cell extracts, stimulated by the presence of DNA. SUMO attachment to K330 affects structural and enzymatic properties of TDG. The modified glycosylase is not longer able to interact with free SUMO or SUMO-conjugated proteins, or to bind stably to AP-sites or any other DNA. Yet, it processes a G-U substrate with enhanced efficiency due to an induced enzymatic turnover but, at the same time, loses its ability to hydrolyze T from a G-T substrate
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SUMO-3
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TDG interacts with, but also is modified by SUMO-1 and SUMO-3, SUMOs are small ubiquitin like modifiers, small polypeptides structurally related to ubiquitin that interact with and/or are attached to other proteins. SUMO conjugation involves Lys330 located in a C-terminal SUMOylation consensus motif, VKEE, it is ATP-dependent and, when performed in cell extracts, stimulated by the presence of DNA. SUMO attachment to K330 affects structural and enzymatic properties of TDG. The modified glycosylase is not longer able to interact with free SUMO or SUMO-conjugated proteins, or to bind stably to AP-sites or any other DNA. Yet, it processes a G-U substrate with enhanced efficiency due to an induced enzymatic turnover but, at the same time, loses its ability to hydrolyze T from a G-T substrate
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000243
3,N4-ethenocytosine-mismatched double-stranded DNA
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excision of 3,N4-ethenocytosine from 3,N4-ethenocytidine/G mismatches
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0.0000128
thymine-mismatched double-stranded DNA
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excision of thymine from T/G mismatches
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0.000012
uracil-mismatched double-stranded DNA
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excision of uracil from U/G mismatches
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additional information
additional information
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pre-steady-state kinetics, and minimal kinetic mechanism for TDG, single turnover kinetics, detailed, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000153
3,N4-ethenocytosine-mismatched double-stranded DNA
Homo sapiens
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excision of 3,N4-ethenocytosine from 3,N4-ethenocytidine/G mismatches
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2.1
5-chlorouracil-mismatched double-stranded DNA
Homo sapiens
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cleavage of 5-chlorouracil from G/5-chlorouracil mismatch
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3.7
5-fluorouracil-mismatched double-stranded DNA
Homo sapiens
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cleavage of 5-fluorouracil from G/5-fluorouracil mismatch
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0.000015 - 0.0036
thymine-mismatched double-stranded DNA
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0.00035 - 0.043
uracil-mismatched double-stranded DNA
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additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
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recombinant His-tagged enzyme
7.5
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assay at
7.8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9.5
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activity range, profile, recombinant His-tagged enzyme, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at
30
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assay at
65 - 70
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recombinant His-tagged enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 85
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activity range, profile, recombinant His-tagged enzyme, overview
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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cellular TDG demonstrates PMA-dependent alterations in apparent molecular weight and isoelectric point
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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proliferating regions of the fetal bone
Manually annotated by BRENDA team
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fetal, high expression
Manually annotated by BRENDA team
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TDG transcripts are uniformly and ubiquitously expressed from 7.5 days post-coitum, but are then markedly enriched in specific tissues of the developing fetus. At 14.5 gestational days, TDG is strongly expressed in the developing nervous system, thymus, lung, liver, kidney and intestine. At later stages, high levels of expression were detected in the thymus, brain, nasal epithelium and within proliferating regions of the intestine, skin, kidney, teeth and bone
Manually annotated by BRENDA team
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proliferating regions of the fetal intestine
Manually annotated by BRENDA team
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proliferating regions of the fetal kidney
Manually annotated by BRENDA team
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fetal, high expression
Manually annotated by BRENDA team
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EC cell
Manually annotated by BRENDA team
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proliferating regions of the fetal skin
Manually annotated by BRENDA team
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proliferating regions of the fetal teeth
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27900
mass spectrometry
46000
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x * 46000, recombinant enzyme, SDS-PAGE
55000
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x * 55000, SDS-PAGE
additional information
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cellular TDG demonstrates PMA-dependent alterations in apparent molecular weight and isoelectric point
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
-
additional information
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primary and domain structure, tertiary structure and structure-function analysis, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetylation
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TDG lysines are acetylated by CREB-binding protein, CBP, and p300. Acetylation of the N-terminal region abrogates high-affinity DNA binding and selectively prevents processing of G:T mispairs. TDG acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG
phosphoprotein
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protein kinase C alpha interacts with TDG and phosphorylates N-terminal serine residues adjacent to lysines acetylated by CREB-binding protein, CBP, and p300. TDG acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG. Phosphorylation does not markedly alter DNA interactions, but may preserve G:T processing in vivo by preventing CBP-mediated acetylation
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of hTDG (catalytic domain, hTDGcat) in complex with abasic DNA, at 2.8 A resolution. The enzyme crystallizes in a 2:1 complex with DNA, one subunit bound at the abasic site, and the other at an undamaged (nonspecific) site
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crystals of SUMO-1–TDG complex are grown in 25% PEG 3350, 0.2 M MgCl2 and 0.1 M Tris-HCl (pH 8.5) at 20°C by using a micro-seeding technique. Crystal structure of the central region of human TDG conjugated to SUMO-1 at 2.1 A resolution
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crystals of SUMO-3–TDG are grown in 1.5 M sodium malonate (pH 5.0) at 20°C, by using a streaking technique. Crystal structure of the central region of TDG conjugated to SUMO-3
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in complex with a 28-base pair DNA containing a guanine:5-hydroxymethyluracil mismatch, hanging drop vapor diffusion method, using 30% (w/v) polyethylene glycol 4000, 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 4.6
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in complex with duplex DNA containing either 5-carboxylcytosine or a 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-carbonylcytosine, hanging drop vapor diffusion method, using
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mutant N140A in complex with guanine:5-carboxylcytosine containing DNA, using 30% (w/v) polyethylene glycol 4000, 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 4.6
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structural basis of substrate specificity
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
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purified recombinant enzyme, 30 min, 50% activity remaining
95
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purified recombinant enzyme, 5 min, inactivation
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA sequence encoding human TDG (residues 112–339) is subcloned into a pGEX4T-3 vector. SUMO-3-modified TDG112–339 (SUMO-3–TDG) is expressed bacterially using an Escherichia coli SUMOylation system by co-transforming the Escherichia coli strain BL21(DE3) with TDG112–339/pGEX4T-3 and pT-E1E2S2 protein expression vectors
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glutathione Sepharose column chromatography
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nickel-charged chelating column chromatography, HiTrap SP column chromatography, and Superdex 75 gel filtration
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recombinant
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recombinant enzyme from NIH3T3 fibroblasts
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recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography
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recombinant His-tagged TDG from Escherichia coli strain BL21 (DE3)
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, phylogenetic tree of MUG proteins, expression in African green monkey kidney cells, the enzyme efficiently replaces the T with a C in G-T mismatched SV40 DNA exhibiting a G-T directed repair activity
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expressed in Escherichia coli BL21(DE3)-Gold cells
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expressed in Escherichia coli Bl21-CodonPlus (DE3)-RP cells
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expression in Escherichia coli
expression in in cell-free extract and in Escherichia coli
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expression in mismatch-transfected B16F10 cells
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expression in NIH3T3 fibroblasts
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expression of the His-tagged enzyme in Escherichia coli strain BL21 (DE3)
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gene Thd1, phylogenetic analysis, functional expression of a His-tagged truncated variant comprising residues 650M-1063N in Escherichia coli strain BL21 (DE3)
HeLa cell clones either stably transfected with a construct overexpressing human TDG from a cytomegalovirus promoter or with the corresponding vector only
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phylogenetic analysis, expression of His-tagged TDG in Escherichia coli strain BL21 (DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
in colorectal cancer patients, enzyme levels are significantly higher in tumor tissues than in the adjacent normal tissues
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wild type p53 binds to a domain of the enzyme promoter containing two p53 consensus response elements and activates its transcription
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D133A
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the mutation significantly reduces the enzyme’s ability to enhance Wnt signaling
N140A/R275L
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site-directed mutagenesis, altered kinetics compared to the wild-type enzyme, overview
N140D
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the mutant exhibits 96fold and 5fold reduced activity at pH 6.0 after 24 h for guanine:uracil and after 72 h for guanine:5-carboxylcytosine, respectively
N140D/N157D
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the mutant exhibits no activity for DNA containing guanine:uracil and guanine:5-carboxylcytosine pairs
N157A
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the mutant exhibits 6fold and 3fold reduced activity at pH 6.0 after 24 h for guanine:uracil and after 72 h for guanine:5-carboxylcytosine, respectively
N157D
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the mutant exhibits 1350fold and 1.1fold reduced activity at pH 6.0 after 24 h for guanine:uracil and after 72 h for guanine:5-carboxylcytosine, respectively
N157D/N230D
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the mutant exhibits 135fold reduced activity and no activity at pH 6.0 after 24 h for guanine:uracil and after 72 h for guanine:5-carboxylcytosine, respectively
N230D
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the mutant exhibits 32fold and 3fold reduced activity at pH 6.0 after 24 h for guanine:uracil and after 72 h for guanine:5-carboxylcytosine, respectively
R275A
-
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme, overview
R275L
-
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme, overview
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
5-fluorouracil is used in clinical cancer therapy. The status of TDG expression in a cancer is likely to determine its response to 5-fluorouracil-based chemotherapy