Information on EC 3.1.25.1 - deoxyribonuclease (pyrimidine dimer)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.1.25.1
-
RECOMMENDED NAME
GeneOntology No.
deoxyribonuclease (pyrimidine dimer)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endonucleolytic cleavage near pyrimidine dimers to products with 5'-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
-
-
hydrolysis of phosphoric ester
CAS REGISTRY NUMBER
COMMENTARY hide
52227-85-7
-
60616-81-1
-
66143-22-4
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
the T4 endonuclease V is a DNA repair enzyme
additional information
-
treatment of Cdk4R24C/R24C/NrasQ61K mice topically with the T4 endonuclease V DNA repair enzyme immediately prior to neonatal ultraviolet radiation exposure has a powerful effect in exacerbating melanoma development in the mouse model, overview. Dimericine reduces the incidence of basal-cell and squamous cell carcinoma. No difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduces DNA damage and cellular proliferation in the skin. Proliferation of melanocytes is inhibited by Dimericine treatment and reduces the level of DNA damage following UVR exposure
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
bacteriophage lambda DNA + H2O
oligonucleotides
show the reaction diagram
-
UV irradiated
-
?
ColE1 DNA + H2O
oligonucleotides
show the reaction diagram
-
UV-irradiated, superhelical
-
?
damaged DNA containing nucleotide cross-links + H2O
?
show the reaction diagram
DNA + H2O
?
show the reaction diagram
DNA containing apurinic/apyrimidinic sites + H2O
?
show the reaction diagram
DNA with a C8 guanine adduct of N-acetyl-2-aminofluorene + H2O
?
show the reaction diagram
-
-
-
-
?
DNA with gamma-radiation-induced cross-linked guanine-thymine tandem lesion G[8,5-Me]T + H2O
?
show the reaction diagram
-
the DNA substrate contains a covalent bond between C8 of the guanine and the 5-methyl carbon of the 3'-thymine, formed by X-radiation
-
-
?
DNA with gamma-radiation-induced cross-linked thymine-thymine lesion T[6,4]T + H2O
?
show the reaction diagram
-
6-4 pyrimidine-pyrimidone photoproduct
-
-
?
dsDNA + H2O
?
show the reaction diagram
-
excision of cyclobutane pyrimidine dimers TT and TU, the latter being a cis-syn cyclobutane thymine-uracil dimer, produced within duplex DNA by UV radiation with equal efficiency, U is derived from C which becomes deaminated by UV radiation
-
-
?
E. coli DNA + H2O
oligonucleotides
show the reaction diagram
E. coli plasmid DNA
oligonucleotides
show the reaction diagram
-
UV-irradiated
-
?
glycosylated DNA + H2O
oligonucleotides
show the reaction diagram
-
UV-irradiated, cleaves also nonglycosylated DNA
-
?
Haemophilus influenzae DNA + H2O
oligonucleotides
show the reaction diagram
-
UV irradiated
-
?
M-13 DNA + H2O
oligonucleotides
show the reaction diagram
-
UV-irradiated, single-stranded DNA
-
?
Micrococcus luteus DNA + H2O
oligonucleotides
show the reaction diagram
-
UV irradiated
-
?
native DNA + H2O
oligonucleotides
show the reaction diagram
oligodeoxyribonucleotides
oligonucleotides
show the reaction diagram
oligonucleotide DNA + H2O
?
show the reaction diagram
pBR322 DNA + H2O
oligonucleotides
show the reaction diagram
PBS1 DNA
oligonucleotides
show the reaction diagram
poly(dA)-poly(dT) + H2O
oligonucleotides
show the reaction diagram
poly(dI)-poly(dC) + H2O
oligonucleotides
show the reaction diagram
-
UV irradiated, in 50 mM phosphate or 10 mM Tris-HCl supplemented with 100 mM NaCl
-
?
SPO1 DNA
oligonucleotides
show the reaction diagram
supercoiled DNA + H2O
?
show the reaction diagram
-
-
-
-
?
superhelical DNA + H2O
oligonucleotides
show the reaction diagram
-
UV-irradiated, cleaves also linear DNA
-
?
T4 DNA
oligonucleotides
show the reaction diagram
-
UV-irradiated
-
?
T7 DNA + H2O
oligonucleotides
show the reaction diagram
-
UV-irradiated
-
?
UV-damaged DNA containing cyclobutane pyrimidine dimers
?
show the reaction diagram
UV-radiation damaged DNA + H2O
?
show the reaction diagram
UV-radiation damaged PM2 phage DNA + H2O
?
show the reaction diagram
X174 DNA + H2O
RFII
show the reaction diagram
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
damaged DNA containing nucleotide cross-links + H2O
?
show the reaction diagram
DNA + H2O
?
show the reaction diagram
supercoiled DNA + H2O
?
show the reaction diagram
-
-
-
-
?
UV-damaged DNA containing cyclobutane pyrimidine dimers
?
show the reaction diagram
-
the T4 enzyme, in so called T4N5 liposomes, repairs UV radiation-induced cyclobutane pyrimidine dimers in DNA of epidermal dendritic cells of human skin which reduces the infiltration of macrophages into irradiated human epidermis and leads to memory immunosuppression in humans, overview
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
potassium phosphate
-
activating, optimum for Py-Py correndonuclease I: 50 mM supplemented with 10 mM Tris-HCl
additional information
-
the enzyme requires a metal ion for activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
-
inhibits both Py-Py correndonucleases
CH2O
-
total inhibition if 4.4 methylations per molecule are present
EDTA
-
inhibits Py-Py correndonuclease II at concentrations above 20 mM
KCl
-
Py-Py correndonuclease II loses 75% of activity in presence of 100 mM
NaBH4
NaCNBH3
-
total inhibition if 4.4 methylations per molecule are present
-
p-chloromercuribenzoate
-
70-80% inhibition in presence of 0.0004 M
p-Chloromercuriphenylsulfonic acid
-
20-25% inhibition in presence of 0.0001 M
Zn2+
-
inhibits both Py-Py correndonucleases
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
rATP
-
stimulates Py-Py correndonuclease I activity
-
Tris-HCl
-
optimum for Py-Py correndonuclease I: 10 mM supplemented with 50 mM NaCl or potassium phosphate; optimum for Py-Py correndonuclease II: 10 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000015 - 0.000019
DNA 14mer containing a TT-dimer
-
0.000009
DNA 18mer containing a TT-dimer
-
-
-
additional information
additional information
-
DNA-enzyme binding kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00833 - 0.02
DNA 14mer containing a TT-dimer
-
0.0183
DNA 18mer containing a TT-dimer
Enterobacteria phage T4
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00063
-
endonuclease from T4 mutant strain F794, partially purified
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
-
pH-optimum of glycosylase activity
6
-
pH-optimum of endonuclease activity
6.5
-
pH-optimum of endonuclease activity
7 - 8
-
pH-optimum of endonuclease activity
7 - 7.4
-
Py-Py correndonucleases I and II
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
-
Py-Py correndonucleases I and II, no activity below or above these values
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
plasmacytoma cell line
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11000 - 15000
-
Py-Py correndonucleases I and II
17120
-
amino acid calculation
17700
-
SDS-PAGE
29310 - 30340
-
cloning identified an open reading frame with two stop codons encoding for two enzymes with these calculated masses
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
-
methylation, after treatment with CH2O and NaCNBH3, N-terminal alpha-amino group is preferentially methylated, glycosylase and endonuclease activity are reduced to 20% if 0.8 methylations are present per enzyme molecule, with 4.4 methyl groups per enzyme both nicking and DNA-binding are abolished
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
monoclinic space group P2-1, 2.3 A resolution
-
purified recombinant enzyme and mutants in complex with DNA containing a thymidine-dimer, X-ray diffraction structure determination and analysis at 1.45-1.6 A and 2.75 A resolution, the latter by molecular replacement method
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
stable at pH 7.0 and above, unstable in acidic solution
170699
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
Py-Py correndonuclease I loses 20% of activity after 5 min, Py-Py correndonuclease II loses more than 75% of activity after 5 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
addition of 8 M urea leads to denaturation
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-methylpentane
-
unstable
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-12C, in 12% glycerol, inactivates the enzyme
-
-20C, addition of an equal value of glycerol
-
-20C, addition of an equal value of glycerol, 1 ml aliquots
-
-20C, in 50% glycerol, complete inactivation in 3-4 weeks
-
-70C, purified enzyme, 20 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.01% Triton X-100, 50 mM KCl, 6 months, loss of 55% activity
4C, in 10 mM Tris-HCl buffer, pH 8.0, with 3% polyethylene glycol and 0.1 mM EDTA
-
4C, in 50 mM Tris-HCl, pH 7.5, containing 10% ethylene glycol or 3% polyethylene glycol, storage for several months
-
4C, stock enzyme-containing cell extracts
-
freezing, leads to inactivation
-
in liquid nitrogen, for minimal inactivation
-
in liquid nitrogen, for minimal inactivation, freeze-thawing does not inactivate either Py-Py correndonuclease
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
100-300fold, extensive purification, attempts to concentrate the enzyme preparation by Aquacide II or Sephadex G50 powder results in loss of activity, UV-DNA-cellulose chromatography, hydroxyapatite chromatography, gel filtration through Sephadex or CM-Sephadex as well as sedimentation in sucrose density gradients results in loss of activity if used after phosphocellulose chromatography, enzyme is more stable in fractions containing large amounts of protein like bovine serum albumin
-
4100fold, to homogeneity
-
native enzyme
-
recombinant enzyme
-
recombinant from Escherichia coli
-
recombinant UvrA, UvrB, and UvrC
-
recombinant wild-type and mutant C-terminally His-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel chelating affinity chromatography to homogeneity
-
to homogeneity
-
WT-enzyme and W128S
-
WT-enzyme: 300fold, enzyme from mutant strain F794: partially purified
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
gene endo V, expression of wild-type and mutant C-terminally His-tagged enzymes in Escherichia coli strain BL21(DE3)
-
overexpression
overexpression in Escherichia coli
-
overexpression of UvrA, UvrB, and UvrC
-
synthetic gene, expressed in Escherichia coli
-
transfected into human repair-proficient fibroblasts, repair deficient xeroderma pigmentosa fibroblasts and wild-type CHO hamster cells
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A26Q
-
not processive
A3D
-
AP-specific nicking possible, low level of dimer specific nicking
A3E
-
AP-specific nicking possible, low level of dimer specific nicking
A3K
-
substantial level of AP- and dimer specific nicking, not processive
A3L
-
substantial level of AP- and dimer specific nicking, not processive
A3Q
-
substantial level of AP- and dimer specific nicking, not processive
DELTAC127
-
C127-truncated, total loss of activity
G133D
-
impaired ability to bind dimer-containing DNA
K130L
-
almost same activity as wild-type
K33Q
-
in combination with A26Q, not processive
R3Q
-
active site mutant, retains full substrate binding ability but shows complex structure differences, crystal structure determination with a bound DNA duplex in comparison to the wild-type enzyme
T127M
-
almost same activity as wild-type, Km value in the same order as that of wild-type
T2P
-
lack of dimer-specific glycosylase and nicking activity
T2S
-
wild-type level of dimer-specific glycosylase and nicking activity
T2V
-
intermediate activity level of dimer-specific glycosylase and nicking activity
W128A
-
total loss of activity
Y129A
-
total loss of activity
Y131A
-
only slight activity remains
Y132A
-
only slight activity remains
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
application of the enzyme packaged in an engineered liposome delivery vehicle to humans suffering the disease xeroderma pigmentosum due to DNA damage by UV radiation reverses the defective repair in these cells, it traverses the stratum corneum, reaches the nuclei of living skin cells, and enhances the repair of UV-induced cyclobutane pyrimidine dimers
molecular biology