Information on EC 3.2.2.17 - deoxyribodipyrimidine endonucleosidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.2.2.17
-
RECOMMENDED NAME
GeneOntology No.
deoxyribodipyrimidine endonucleosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cleaves the N-glycosidic bond between the 5'-pyrimidine residue in cyclobutadipyrimidine (in DNA) and the corresponding deoxy-D-ribose residue
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of N-glycosyl bond
SYSTEMATIC NAME
IUBMB Comments
deoxy-D-ribocyclobutadipyrimidine polynucleotidodeoxyribohydrolase
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
16-mers oligodeoxynucleotide + H2O
?
show the reaction diagram
-
anomers of methyl-deoxyribofuranose analogues indicates that T4-pdg preferentially cleaved the beta-anomers
-
-
?
DNA + H2O
?
show the reaction diagram
DNA + H2O
thymine + ?
show the reaction diagram
DNA phiX174 RFI + H2O
thymine + ?
show the reaction diagram
oligonucleotide + H2O
?
show the reaction diagram
oligonucleotides + H2O
dCMP + dTMP
show the reaction diagram
poly(dAdT) + H2O
oligonucleotides
show the reaction diagram
-
-
acid-soluble oligonucleotides, length less than 15 nucleotides
?
additional information
?
-
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
DNA + H2O
?
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
-
slightly more stimulatory than NaCl
MgCl2
-
catalytic activity 7 times greater than in presence of EDTA, DNA glycosylase activity preceded by a Mg2+ dependent AP endonuclease, followed by an AP endonuclease active in the absence of Mg2+ and in presence of EDTA; not stimulated by CaCl2, MnCl2, ZnCl2; unaffected by the presence of ATP
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-[N-(acetoxyacetyl)amino]fluorene
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AAAF, carcinogen , extensive modification of DNA does not significantly reduce the thymine dimer yield
DNA
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depurinated DNA competitively inhibits the UV endonuclease activity, irradiated DNA inhibits the AP endonuclease activity
NaBH4
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
isopropyl-1-thio-beta-D-galactopyranoside
-
induction of the pdg gene
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00000014 - 0.002556
DNA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 37
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17500
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 26000, SDS-PAGE and gel filtration
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 amino acids critical for T4-PDG catalysis, Thr2 and Glu23, known from x-ray crystal structure studies
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crystallized at 15°C by the hanging-drop vapor-diffusion method, co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (about 66°) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42
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AP endonuclease destroyed very rapidly, glycosylase activity lost at linear rate of 4% per min
43
-
no significant reduction in activity when incubated for 2 min at 43°C
45 - 55
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thermal inactivation of both endonuclease and glycosylase activity
46
-
2 enzyme activities resides in the same protein molecule, activities lost in parallel during incubation for up to 17 min at 46°C
50
-
UV endonuclease and AP endonuclease inactivated in parallel at 50°C
90
-
thermostable, difficult to completely heat inactivate, retains 10% activity after 20 min at 90°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
relatively stable and soluble under most conditions
-
resistant to EDTA
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C stable when stored in 50% glycerol, -20°C stable for several freeze-thaw cycles, 4°C in buffer without glycerol for up to a year without loss of activity
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4°C, pH 6.8, for several days with no loss of activity
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stable on ice for 8 months with no loss in activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cv-pdg mutants T2S, T2S/E23D, T2P, T'P/E23D
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purified from Escherichia coli AB2480
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site-directed mutant enzymes
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the recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria is purified using one-step affinity chromatography with a HiTrap Chelating HP column
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to homogeinity, using several chromatographic steps
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to homogeneity, using several chromatographic steps
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to pure protein (~ 20 mg/ml) using buffer solutions and column filled of chitin matrix
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned and sequenced from 42 Chlorella viruses isolated over 12 years in diverse geographic regions
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cloned into a variety of plasmids, express endonuclease V activity in repair-deficient Escherichia coli
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cloned into the pTYB2 vector and overexpressed in Escherichia coli
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denVgene cloned and sequenced
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expression in Escherichia coli of several mutants
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gene cv-pdg encoding T4 endonucleaseV homolog, 41% identical to T4endoV, enhance UV radiation resistance in repair-deficient Escherichia coli BL21, vector pCRII, cloning and overexpression of the gene product, cloned into the Escherichia coli expression plasmid pET11a
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PCR-amplified from pET11a, subcloned into pTYB2, plasmids subsequently transformed into Escherichia coli ER2566 for expression
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PCR-amplified from pUC19, subcloned into pET-11a
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pEVC127, cloned and expressed in Escherichia coli HB101, synthetic gene constructed by ligation of oligonucleotide fragments expressed efficiently in Escherichia coli
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T4 denV (414 bp cDNA) encoding T4 endo V (138 amino acid) is synthesized and expressed using either an expression vector, pTriEx-4, in Escherichia coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H16A
-
decrease in enzymic activity, analysis of initial velocity
H16C
-
decrease in enzymic activity, analysis of initial velocity
H16D
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decrease in enzymic activity, analysis of initial velocity
H16E
-
decrease in enzymic activity, analysis of initial velocity
H16K
-
decrease in enzymic activity, analysis of initial velocity
H16S
-
decrease in enzymic activity, analysis of initial velocity
H16A
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
H16C
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
H16D
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
H16E
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
H16K
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
H16S
-
performs most functions tested in vivo, although at reduced rates compared with the wild type protein
additional information
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treatment of Cdk4(R24C/R24C)/Nras(Q61K) mice topically with the T4 endonuclease V DNA repair enzyme immediately prior to neonatal ultraviolet radiation (UVR) exposure has a powerful effect in exacerbating melanoma development, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
medicine
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topical DNA repair enzyme application may be a clinically useful approach of photo-protection in humans. The most useful role for T4 endonuclease V would be its inclusion in sun-block