Information on EC 3.1.21.7 - deoxyribonuclease V

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.1.21.7
-
RECOMMENDED NAME
GeneOntology No.
deoxyribonuclease V
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endonucleolytic cleavage at apurinic or apyrimidinic sites to products with a 5'-phosphate
show the reaction diagram
Previously classified erroneously as EC 3.1.22.3
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric diester
-
-
hydrolysis of phosphoric ester
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
61970-03-4
Previously classified erroneously as EC 3.1.22.3
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
this DNA repair enzyme is also involved in RNA metabolism
physiological function
additional information
-
the deoxyinosine-containing heteroduplex is added to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. The three proteins alone are sufficient to process the dI lesion efficiently, and the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
12-mer oligonucleotide + H2O
hydrolyzed DNA
show the reaction diagram
-
double-stranded, containing a site-specific duplex cis-syn cyclobutane pyrimidine dimer
-
?
34-mer oligonucleotide + H2O
fragments of 34mer-oligonucleotide
show the reaction diagram
-
oligonucleotide duplex containing a fluorine atom at the 2' position of the 5' component of the cyclobutane pyrimidine dimer
-
-
?
48-mer oligonucleotide + H2O
24-mer oligonucleotide
show the reaction diagram
-
double-stranded, containing a site-specific duplex cis-syn cyclobutane pyrimidine dimer
2,3-didehydro-2,3-dideoxyribose 5'-phosphate at the 3'-terminus of the products, initiation of repair synthesis by E. coli DNA-polymerase I
?
49-mer oligonucleotide + H2O
3-alpha,beta-unsaturated aldehyde + 3'-phosphate DNA-termini
show the reaction diagram
-
double-stranded substrate, containing a site-specific duplex cis-syn cyclobutane pyrimidine dimer, enzyme requires a secondary binding event after beta-elimination at a pyrimidine dimer or abasic site
delta-elimination reaction
?
49-mer oligonucleotide + H2O
hydrolysed DNA
show the reaction diagram
deoxyinosine-containing deoxyoligonucleotide + H2O
?
show the reaction diagram
deoxyinosine-containing single-stranded DNA + H2O
?
show the reaction diagram
-
precursor activity of DNA repair
-
-
?
DNA + H2O
3-alpha,beta-unsaturated aldehyde + 5'-phosphate DNA-termini
show the reaction diagram
-
abasic lyase activity
-
?
DNA + H2O
5'-phosphoryltermini DNA + 3'-hydroxyltermini DNA
show the reaction diagram
DNA + H2O
?
show the reaction diagram
DNA + H2O
hydrolysed DNA
show the reaction diagram
double-stranded plasmid DNA + H2O
nicked circular DNA + linearized DNA
show the reaction diagram
dsDNA containing deoxyinosine + H2O
?
show the reaction diagram
no cleavage activity is detected with the dsDNA containing a T/G mismatch base pair and a dsDNA containing an apurinic site. The enzyme possesses strict substrate specificity to deoxyinosine-containing DNA. The enzyme cleaves inosine-containing RNA strands as well as DNA substrates
-
-
?
nicked DNA + H2O
?
show the reaction diagram
-
-
-
-
?
RNA + H2O
?
show the reaction diagram
-
the enzyme also cleaves inosine-containing RNA strands
-
-
?
RNA-DNA hybrid + H2O
?
show the reaction diagram
single-stranded circular DNA + H2O
linearized single-stranded DNA
show the reaction diagram
-
10fold more activity than for duplex DNA
-
?
single-stranded RNA + H2O
?
show the reaction diagram
5'-CUGACUICGGAUCAGGGCC-3'. The enzyme is most active towards single-stranded RNA but is much less active towards other substrates
-
-
?
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
deoxyinosine-containing deoxyoligonucleotide + H2O
?
show the reaction diagram
-
the enzyme is involved in damaged DNA repair
-
-
?
DNA + H2O
5'-phosphoryltermini DNA + 3'-hydroxyltermini DNA
show the reaction diagram
DNA + H2O
?
show the reaction diagram
double-stranded plasmid DNA + H2O
nicked circular DNA + linearized DNA
show the reaction diagram
nicked DNA + H2O
?
show the reaction diagram
-
-
-
-
?
RNA + H2O
?
show the reaction diagram
-
the enzyme also cleaves inosine-containing RNA strands
-
-
?
RNA-DNA hybrid + H2O
?
show the reaction diagram
single-stranded circular DNA + H2O
linearized single-stranded DNA
show the reaction diagram
-
10fold more activity than for duplex DNA
-
?
single-stranded RNA + H2O
?
show the reaction diagram
Q8N8Q3
5'-CUGACUICGGAUCAGGGCC-3'. The enzyme is most active towards single-stranded RNA but is much less active towards other substrates
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
supports oxanosine-containing DNA cleavage to a small extent
Ni2+
-
supports oxanosine-containing DNA cleavage to a small extent
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
50% inhibition at 25 mM, inhibition of formation of acid-soluble products
Ag2+
-
50% inhibition at 0.01 mM
Cu2+
-
50% inhibition at 0.025 mM
Fe3+
-
strong inhibition
Hg2+
-
50% inhibition at 0.0006 mM, inhibition of both DNA glycosylase and apurinic nicking activities, binding capability not reduced
NaCN
-
50% inhibition at 3-5 mM
tRNA
-
half-maximal inhibition rate at 0.08 mM
additional information
-
an oligonucleotide duplex containing a 2'-fluorinated sugar moiety at the cyclobutane pyrimidine dimer (CPD) site inhibits the T4 Endo V reaction by stabilizing the covalent enzyme-DNA complex
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
-
required
additional information
-
high pH, metal cofactor Mn2+, using excess enzyme, and/or solvents such as dimethyl sulfoxide and betaine, lead to cleavage of most mismatched DNA base pairs
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003 - 0.024
49-mer oligonucleotide
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000333 - 0.0117
49-mer oligonucleotide
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.52
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6
-
-
7 - 7.5
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activity for deoxyinosine-containing DNA
7.6
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endonuclease V incision assay
7.9
-
reconstituted endonuclease V-mediated excision repair assay at
8 - 8.5
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activity for deoxyuridine-containing DNA
8
-
mismatch repair activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.8
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pH 7.0: about 45% of maximal activity, pH 8.8: about 90% of maximal activity
7.4
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
a moderate reaction temperature is used for the biochemical studies, because of the melting temperature of the DNA duplex substrates (Tm = 67ºC)
65
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 100
-
60°C: about 45% of maximal activity, 100°C: about 70% of maximal activity
70 - 80
-
70°C: about 50% of maximal activity, 80°C: about 35% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.8
-
calculated from sequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Escherichia coli O45:K1 (strain S88 / ExPEC)
Streptomyces avermitilis (strain ATCC 31267 / DSM 46492 / JCM 5070 / NBRC 14893 / NCIMB 12804 / NRRL 8165 / MA-4680)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
-
2 * 10000, SDS-PAGE, in the presence of 100 mM KCl
13000
-
4 * 13000, SDS-PAGE
19000
-
gel filtration in the presence of 100 mM KCl
20000
-
glycerol density gradient sedimentation
23580
-
x * 23580, calculated from sequence, His-tagged enzyme
27000
-
glycerol density gradient centrifugation
27800
-
1 * 27800, calculated from amino acid sequence
28800
-
1 * 28800, SDS-PAGE
53000
-
sucrose density gradient centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 23580, calculated from sequence, His-tagged enzyme
dimer
-
2 * 10000, SDS-PAGE, in the presence of 100 mM KCl
monomer
tetramer
-
4 * 13000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2.5 A resolution
-
2.75 A resolution, complexed with a duplex DNA substrate, adenine base complementary to the 5'-side of the thymine dimer is completely flipped out of the DNA duplex
-
sitting drop vapor diffusion method, using 19% (w/v) PEG 3350, 0.2 M sodium formate, 0.1 M Tris-HCl pH 7.5 or 1.4 M ammonium sulfate, 0.2 M sodium acetate, 0.1 M Tris-HCl pH 7.5
-
EndoV in complex with a hypoxanthine lesion substrate and with product DNA, hanging drop vapour diffusion method, in 6-12% (w/v) MPEG 2k in 200 mM imidazole-matate buffer, pH 5.8-7.4
-
in complex with DNA containing one nucleotide A:TT loop, vapor diffusion method, using 15% (w/v) polyethylene glycol 400 and 100 mM MES, pH 6.3
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
50% remaining activity
288788
9
-
30% remaining activity for deoxyinosine-containing DNA above, less than 10% remaining mismatch repair activity above
288775
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable to freeze-thawing, unstable for long-term storage
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50% glycerol, somewhat stable for several months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chromatography techniques
-
HiTrap SP column chromatography, and Superdex 75 gel filtration
-
HiTrap SP XL column chromatography
-
Ni-NTA column chromatography and Superdex 75 gel filtration
-
Ni-NTA column chromatography, HiTrap heparin column chromatography, and Superdex 200 gel filtration
-
to homogeneity, chromatography steps
-
to homogeneity, chromatography steps, recombinant enzymes
-
to homogeneity, chromatography techniques
-
to homogeneity, recombinant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli BL21-Codon Plus (DE3)-RIPL cells
-
expressed in Escherichia coli strain BL21 CodonPlus (DE3) RIL
-
expression in Escherichia coli
expression in Escherichia coli GWR109
-
overexpression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C78T
-
100 times more activity in the presence Hg2+ or Ag+ at concentrations they are required to inhibit the wild type enzyme
E23D
-
altered affinities for pyrrolidine- and tetrahydrofuran-residues or reduced apurinic sites
F59L
-
remains in the monomeric state independent of salt concentration, alteration of target site location
F60L
-
remains in the monomeric state independent of salt concentration, alteration of target site location
K121N
-
decreased affinity for nontarget DNA
K86N
-
decreased affinity for nontarget DNA
Q15R
-
limited processive nicking activity
R117N
-
limited processive nickin activity
R3Q
-
greatly decreased DNA binding ability
D35A
-
inactive
D52A
-
inactive
Y91A
-
the mutant has lost its affinity for branched DNA substrates
D35A
-
inactive mutant enzyme
A123I
-
levels of oxanosine and uridine cleavage are reduced by more than 90%
A138I
-
mutation reduces level of T/I cleavage by 10%
A86M
-
fully active in inosine and xanthosine substrates, significant loss in the level of oxanosine and uridine cleavage
D110A
-
little cleavage activity
D110C
-
mutant
D110E
-
mutant
D110H
-
mutant
D43C
-
no more than 10% cleavage activity
D43E
-
no more than 10% cleavage activity
D43H
-
no more than 10% cleavage activity
E89A
-
about 20-30% cleavage activity
E89D
-
mutant
E89H
-
mutant
F46A
-
mutation reduces the levels of oxanosine and uridine cleavage to less than 40%
F87A
-
mutant essentially maintains wild-type level activity towards inosine, xanthosine, oxanosine and uridine substrates
G111V
-
levels of oxanosine and uridine cleavage are reduced by more than 90%, level of cleavage of the T/I substrate is reduced by 40%
G113V
-
levels of oxanosine and uridine cleavage are reduced by more than 90%, level of cleavage of the T/I substrate is reduced by 50%
G121V
-
levels of oxanosine and uridine cleavage are reduced by more than 90%, level of cleavage of the T/I substrate is reduced by 10%
G127V
-
levels of oxanosine and uridine cleavage are reduced by more than 90%, level of cleavage of the T/I substrate is reduced by 70%
G184V
-
mutation reduces the level of inosine and xanthosine cleavage
G83V
-
fully active in inosine and xanthosine substrates, significant loss in the level of oxanosine and uridine cleavage
H116T
-
DNA and Mn2+ binding defective enzyme mutant
H214A
-
tolerant of mutation without impacting activity
H214C
-
tolerant of mutation without impacting activity
K139A
-
mutation reduces level of T/I cleavage by 10%
K139Q
-
mutation reduces level of T/I cleavage by 10%
K139R
-
mutation reduces level of T/I cleavage by 10%
L85V
-
fully active in inosine and xanthosine substrates, significant loss in the level of oxanosine and uridine cleavage
P79A
-
mutant essentially maintains wild-type level activity towards inosine, xanthosine, oxanosine and uridine substrates
Q20A
-
DNA and Mn2+ binding defective enzyme mutant
R88K
-
mismatch cleavage activity similar to the wild-type enzyme
R88Q
-
mismatch cleavage activity similar to the wild-type enzyme
R99Q
-
fully active in inosine and xanthosine substrates, significant loss in the level of oxanosine and uridine cleavage
V137A
-
mutation reduces level of T/I cleavage by 10%
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
determination of the minimum value of triplet energies for a photosensitizer to produce thymine cyclobutane dimer in DNA
biotechnology
-
method of linear amplification of DNA, allows 100fold amplification of target molecules
drug development
-
investigated as a chemopreventive agent of nonmelanoma skin cancer
medicine
-
in xeroderma pigmentosum patients, topical application of liposome-encapsulated T4 endonuclease V reduces the incidence of basal cell carcinomas by 30% and of actinic keratoses by more than 68%
molecular biology
-
application to DNA shuffling. Random DNA fragmentation by endonuclease V is a handy and reproducible method and can be used instead of fragmentation by DNase I, which is technically problematic due to decreasing shuffling efficiency
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