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Enzyme Nomenclature
Information on EC 3.2.2.28 - double-stranded uracil-DNA glycosylase
Word Map on EC 3.2.2.28
- 3.2.2.28
-
excision
- thymine
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thymine-dna
- cytosine
- guanine
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mispairs
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deamination
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n2,3-ethenoguanine
- purine
- aldehyde
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isogenic
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thermus
- 3,n4-ethenocytosine
- pyrimidine
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single-stranded
- dutp
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exocyclic
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pollutants
- thermophilus
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etheno
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
3.2.2.28
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RECOMMENDED NAME
GeneOntology No.
double-stranded uracil-DNA glycosylase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Specifically hydrolyses mismatched double-stranded DNA and polynucleotides, releasing free uracil
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SYSTEMATIC NAME
IUBMB Comments
uracil-double-stranded DNA deoxyribohydrolase (uracil-releasing)
No activity on DNA containing a T/G mispair or single-stranded DNA containing either a site-specific uracil or 3,N4-ethenocytosine residue [2], significant role for double-stranded uracil-DNA glycosylase in mutation avoidance in non-dividing E. coli [3]. Uracil-DNA glycosylases are widespread enzymes that are found in all living organisms. Uracil-DNA glycosylase (EC 3.2.2.27) and EC 3.2.2.28 form a central part of the DNA-repair machinery since they initiate the DNA base-excision repair pathway by hydrolysing the N-glycosidic bond between uracil and the deoxyribose sugar thereby catalysing the removal of mis-incorporated uracil from DNA.
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CAS REGISTRY NUMBER
COMMENTARY
59088-21-0
cf. EC 3.2.2.27
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
3,N4-ethenocytosine-containing single-stranded DNA + H2O
3,N4-ethenocytosine + single-stranded DNA with abasic site
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the enzyme excised both 3,N4-ethenocytosine and uracil from DNA. 3,N4-ethenocytosine is significantly better as a substrate in terms of binding and hydrolysis. The tighter binding of the 3,N4-ethenocytosine containing substrate by MUG probably also accounts for its activity against single-stranded DNA containing 3,N4-ethenocytosine. Cleavage of the single-stranded substrate is 1500fold slower than the double-stranded substrate
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-
?
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
hypoxanthine-mismatched double-stranded DNA + H2O
hypoxanthine + double-stranded DNA with abasic site
the enzyme also acts as a hypoxanthine DNA glycosylase with the strongest activity on the G/I base pair but no activity detected on the C/I base pair
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-
?
xanthine-mismatched double-stranded DNA + H2O
xanthine + double-stranded DNA with abasic site
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-
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?
xanthine-mismatched single-stranded DNA + H2O
xanthine + single-stranded DNA with abasic site
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-
-
?
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
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-
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?
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
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3,N4-ethenocytosine is recognized and efficiently excised from the 3,N4-ethenocytosine/G mismatch
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?
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
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Dug is active on duplex oligonucleotides (34-mers) that contain site-specific 3,N4-ethenocytosine/G, and 3,N4-ethenocytosine/A mismatches
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?
3,N4-ethenocytosine-mismatched double-stranded DNA + H2O
3,N4-ethenocytosine + double-stranded DNA with abasic site
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the enzyme excised both 3,N4-ethenocytosine and uracil from DNA. 3,N4-ethenocytosine is significantly better as a substrate in terms of binding and hydrolysis. The tighter binding of the 3,N4-ethenocytosine containing substrate by MUG probably also accounts for its activity against single-stranded DNA containing 3,N4-ethenocytosine. Cleavage of the single-stranded substrate is 1500fold slower than the double-stranded substrate. Of the different substrates tested, a duplex containing the 3,N4-ethenocytosine pair has the highest affinity for the enzyme, U/G is the next best substrate
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?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
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dsUDG can remove uracil from G/U mispairs
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?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
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Dug is active on duplex oligonucleotides (34-mers) that contain site-specific U/G or U/A mismatches. Dug excises a near stoichiometric amount of uracil from U/G-containing oligonucleotide substrate. The lack of turnover is the result of strong binding by Dug to the reaction product apyrimidinic-site
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?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
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excision of uracil from U/G mismatch
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?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
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the enzyme activity against the uracil-containing single-stranded DNA is so low that it is not likely to be of any significance. Of the different substrates tested, a duplex containing the 3,N4-ethenocytosine pair has the highest affinity for the enzyme, U/G is the next best substrate
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?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
the enzyme acts as a double-stranded uracil DNA glycosylase with a relatively low activity on the A/U base pair
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?
additional information
?
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Mug is expressed poorly in exponentially growing cells and has no apparent role in mutation avoidance in these cells. Mug is fairly abundant in stationary-phase cells and has an important anti-mutator role at this stage of cell growth
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additional information
?
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5-Hydroxyuracil and inosine (hypoxanthine) show cleavage rates 2–3 orders of magnitude slower than 3,N4-ethenocytosine. Thymine, 5-hydroxymethyluracil, and 5-hydroxycytosine are cleaved to some extent, although extremely slowly
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additional information
?
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activity is not detected on DNA containing a T/G mispair or single-stranded DNA containing either a site-specific uracil or 3,N4-ethenocytosine residue. Endonuclease IV stimulates Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the apyrimidinic-site/G-containing 34-mer. Catalytically active endonuclease IV is required to mediate Dug turnover
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additional information
?
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the excision of thymine in a G/T mismatch by dsUDG is extremely low. This enzymatic activity does not have a real biological significance
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additional information
?
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UdgB substrate specificity with diverse DNA oligomers, overview, the enzyme excises ethenocytosine and hypoxanthine from dsDNA, in addition to uracil present as a single-nucleotide bulge in dsDNA, but excision of 5-OH-C, dihydroxyuracil, and epsilonA is undetectable, MtuUdgB does not excise uracil from SSU9 ssDNA, mechanism of action, overview
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additional information
?
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the enzyme showed no activity on oxanine-containing DNA substrates, uracil-mismatched single-stranded DNA and hypoxanthine-mismatched single-stranded DNA
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
hypoxanthine-mismatched double-stranded DNA + H2O
hypoxanthine + double-stranded DNA with abasic site
Q5SJ65
the enzyme also acts as a hypoxanthine DNA glycosylase with the strongest activity on the G/I base pair but no activity detected on the C/I base pair
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-
?
uracil-mismatched double-stranded DNA + H2O
uracil + double-stranded DNA with abasic site
Q5SJ65
the enzyme acts as a double-stranded uracil DNA glycosylase with a relatively low activity on the A/U base pair
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?
xanthine-mismatched double-stranded DNA + H2O
xanthine + double-stranded DNA with abasic site
Q5SJ65
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?
xanthine-mismatched single-stranded DNA + H2O
xanthine + single-stranded DNA with abasic site
Q5SJ65
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?
additional information
?
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Mug is expressed poorly in exponentially growing cells and has no apparent role in mutation avoidance in these cells. Mug is fairly abundant in stationary-phase cells and has an important anti-mutator role at this stage of cell growth
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
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double-strand uracil-DNA glycosylase is insensitive to uracil-DNA glycosylase inhibitor protein, i.e. Ugi
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additional information
UdgB inhibition by dsDNA containing AP-site in the uracil-containing single-nucleotide bulge, UgdB is insensitive against UDG inhibition protein from Bacillus subtilis and shows no inhibition by uracil
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
endonuclease IV
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endonuclease IV stimulates Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the apyrimidinic-site/G-containing 34-mer. Catalytically active endonuclease IV is required to mediate Dug turnover
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
poorly expressed in exponentially growing cells. Mug is overexpressed as cells enter stationary phase, and it is maintained at a fairly high level in resting cells. This is true of cells grown in rich or minimal media, and the principal regulation of mug is at the level of mRNA. Although the expression of mug is strongly dependent on the stationary-phase sigma factor, when cells are grown in minimal media, it shows only a modest dependence on stationary-phase sigma factor when cells are grown in rich media
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PDB
SCOP
CATH
ORGANISM
UNIPROT
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine
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crystal structures of the mismatch-specific uracil DNA-glycosylase from Escherichia coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strains by nickel affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene Rv1259, DNA and amino aid sequence determination and analysis, cloning and overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strains
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D75A
the mutant loses xanthine DNA glycosylase activity while retaining partial uracil and hypoxanthine DNA glycosylase activity
D75N
the mutant loses xanthine DNA glycosylase activity while retaining much of their uracil and hypoxanthine DNA glycosylase activity
D75Q
the mutant loses xanthine DNA glycosylase activity while retaining much of their uracil and hypoxanthine DNA glycosylase activity
N120A
the mutant loses xanthine DNA glycosylase activity while retaining partial uracil and hypoxanthine DNA glycosylase activity
additional information
construction of mutants by insertion of kanamycin resistance marker in the UdgB active site motif GQDPY, phenotypes, overview
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LINKS TO OTHER DATABASES (specific for EC-Number 3.2.2.28)
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