Information on EC 3.1.21.3 - type I site-specific deoxyribonuclease

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

EC NUMBER
COMMENTARY hide
3.1.21.3
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RECOMMENDED NAME
GeneOntology No.
type I site-specific deoxyribonuclease
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endonucleolytic cleavage of DNA to give random double-stranded fragments with terminal 5'-phosphates; ATP is simultaneously hydrolysed
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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CAS REGISTRY NUMBER
COMMENTARY hide
37263-09-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
enzyme CfrAI
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-
Manually annotated by BRENDA team
Escherichia coli A0 34/86 (O83:K24:H31)
strain CPW11
UniProt
Manually annotated by BRENDA team
strain CPW11
UniProt
Manually annotated by BRENDA team
strains 26695, J99, HPAG1, NSH57and NSH79
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-
Manually annotated by BRENDA team
type I restriction-modification enzyme subunit S; GM236
SwissProt
Manually annotated by BRENDA team
type I restriction-modification enzyme subunit S; GM236
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
several strains isolated from infected potato plant. Presence of enzyme Pca17AI, an isoschizomer of EcoRII endonuclease is only observed in isolates of Pectobacterium carotovorum atrospeticum, not in Pectobacterium carotovorum carotovorum
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
enzyme s StySBI, StySJI, StySPI and enzyme StySQI
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-
Manually annotated by BRENDA team
Salmonella-Escherichia coli hybrid
strains L4029 and L4039
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-
Manually annotated by BRENDA team
strain RN450
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-
Manually annotated by BRENDA team
synthetic construct
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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EcoprrI is an apoptosis enzyme
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3'-biotinylated 51 bp dsDNA + H2O
?
show the reaction diagram
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-
-
?
5'-biotinylated 51 bp ssDNA + H2O
?
show the reaction diagram
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-
-
?
DNA + H2O
?
show the reaction diagram
duplex DNA + ATP
double-stranded DNA fragments with terminal 5'-phosphate + ADP + inorganic phosphate
show the reaction diagram
duplex DNA + ATP
double-stranded DNA fragments with terminal 5'-phosphate + ADP + phosphate
show the reaction diagram
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-
-
?
linear plasmid DNA pDRM-2R + ATP
?
show the reaction diagram
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-
-
?
plasmid DNA + H2O
?
show the reaction diagram
plasmid pACYC184 + ATP
?
show the reaction diagram
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-
-
-
?
plasmid pET20b + ATP
?
show the reaction diagram
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-
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?
plasmid pTK-neo + ATP
?
show the reaction diagram
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the enzyme recognises the symmetrical sequence GAAN7TTC at position 2535 bp
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-
?
plasmid pUC19 + ATP
?
show the reaction diagram
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the enzyme recognises the symmetrical sequence GAAN7TTC at positions 1126 bp and 2294 bp
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?
supercoiled plasmid DNA pRK + ATP
?
show the reaction diagram
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-
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?
synthetic oligonucleotide + ATP
?
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
DNA + H2O
?
show the reaction diagram
duplex DNA + ATP
double-stranded DNA fragments with terminal 5'-phosphate + ADP + inorganic phosphate
show the reaction diagram
duplex DNA + ATP
double-stranded DNA fragments with terminal 5'-phosphate + ADP + phosphate
show the reaction diagram
Q7MPU6
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-
-
?
linear plasmid DNA pDRM-2R + ATP
?
show the reaction diagram
P10486
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-
-
?
plasmid DNA + H2O
?
show the reaction diagram
supercoiled plasmid DNA pRK + ATP
?
show the reaction diagram
P10486
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-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ArdA
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antirestriction protein of InvB plasmid R16, selective
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ArdA protein
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ArdB protein
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DNA
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cleavage of DNA is inhibited by an increased degree of negative supercoiling
Ocr protein
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S-adenosyl homocysteine
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competitive
additional information
synthetic construct
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a repressing complex of C.Esp1396I tetramer bound to both binding sites
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl methionine
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stimulates DNA cleavage activity
S-adenosyl-L-methionine
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000015
3'-biotinylated 51 bp dsDNA
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pH 9.4, 30C
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0.000011
5'-biotinylated 51 bp ssDNA
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pH 9.4, 30C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
167
ATP
Escherichia coli
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at 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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EcoB, methylase activity
8
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nuclease reaction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
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optimal temperature for DNA cleavage. Optimal flipping occurs at temperatures substantially below the growth temperature of the source organism for PspGI and for the catalytic activity of endonuclease
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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individual subunits HsdR and HsdM are soluble cytoplasmic proteins
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17920
synthetic construct
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sedimentation equilibrium analytical ultracentrifugation of C.Esp1396I
18450
synthetic construct
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theoretical Mr of C.Esp1396I
290000 - 315000
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pentameric enzyme form R2M2S1, gel filtration
312000
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gel filtration
400000
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EcoK, sucrose density gradient sedimentation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
synthetic construct
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sedimentation equilibrium analytical ultracentrifugation of C.Esp1396I
monomer
1 * 94000, dynamic light scattering, 1 * 130000, analytical ultracentrifugation, subunit HsdR, subunit is globular and fairly compact
octamer
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alpha2,beta4,gamma2, 2 * 135000 + 4 * 60000 + 2 * 55000, EcoB, SDS-PAGE
pentamer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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HsdR subunit of isoform EcoKI is phosphorylated on Thr. Phosphorylation in vitro is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from Escherichia coli. Phosphorylation in vivo only occurs when subunit HsdR is coproduced with subunits HsdM and HsdS
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
database information: http://rebase.neb.com
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model of subunit HsdR using protein fold-recognition and homology modeling. Subunit shows an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains. Conformational heterogenity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex
motor subunit HsdR of pR124 plasmid-borne type I restriction-modification enzyme EcoR124I, solved in complex with Mg2+-ATP at 2.6 A resolution. HsdR presents four globular domains in a square-planar arrangement, generating prominent grooves between adjacent domain pairs. Lys220 on alpha8 is 3.1 A from N3 on the exposed edge of ATP bound at the helicase domains, potentially coupling endonuclease and helicase function. A uniformly positive surface groove with a clear match to the size and shape of duplex DNA proceeds from a canonical helicase cleft in a continuous path down the front of the motor subunit between the helical and endonuclease domains, where the cleavage site is recessed slightly from the surface
recombinant motor subunit HsdR of isoform EcoR124I in presence and absence of ATP, at 2.6 A resolution
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hanging drop vapor diffusion method, using 22% (w/v) polyethylene glycol 8000, 0.02 M imidazole pH 7.5, 5 mM beta-mercaptoethanol
hanging-drop and sitting-drop vapour-diffusion method, HsdR subunit crystallized from 8%(w/v) polyethylene glycol 3350, 0.15 M ammonium chloride, 0.1 M HEPES pH 7.5 and 2 mM beta-mercaptoethanol, to 2.60 A resolution. Crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.01, b = 89.04, c = 113.66 A. With one HsdR molecule in the asymmetric unit, the Matthews coefficient is 2.14 A3 Da-1 and the solvent content is 42%
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HsdM subunit, to 1.86 A resolution, by hanging-drop and sitting-drop vapour-diffusion methods. Crystal belongs to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 78.9, c = 165.8 A. With one molecule in the asymmetric unit, the crystal volume per unit protein weight is 2.12 A3/Da, with a solvent content of 42%
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N-terminal fragment (ca. 590 amino acids) of a putative HsdR subunit (817 amino acids), native crystals and Se-Met crystals, to 2.5 A resolution, by hanging-drop vapor-diffusion method at 22C. Se-Met crystal belongs to the orthorhombic space group P212121 with unit-cell dimensions of a = 71.01, b = 89.04 and c = 113.66 A. Seven Se sites in the asymmetric unit at 3.0 A resolution. Crystal structure reveals catalytic sites for nuclease (NTD-domain, Ala21-Ile159) and ATPase (RecA-like domains: RD1 for Lys160-Tyr360 and RD2 for Ala372-Val590) activities and suggests a possible translocation mechanism of the HsdR subunit
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
C.Esp1396I-6His and C.Esp1396I without hexahistidine tag
synthetic construct
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enzyme EcoB; enzyme EcoK
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HisTrap column chromatography, DEAE column chromatography, and heparin column chromatography
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HsdM subunit, on anion-exchange column, more than 95% pure
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HsdR fusion protein purified by gel filtration. HsdR with five additional amino acids (YFQGA) at the N-terminus purified on anion-exchange column and by gel filtration, purified protein is more than 95% pure
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mixture of two enzyme species, the larger species has the stoichiometry R2M2S1, the smaller species has the stoichiometry of R1M2S1.only the R2M2S1 complex is capable of DNA cleavage, the R1M2S1 complex retains ATPase activity
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Mtase(DELTA50)
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mutant methylase
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native HsdR protein homogeneously purified by sequential chromatographic steps
nickel-affinity column chromatography, ion-exchange chromatography, and gel filtration
recombinant motor subunit HsdR of isoform EcoR124I
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wild-type R.PspGI and its mutant purified by chromatography, to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
248-bp insertion fragments of the ptypeI plasmid inserted at the HincII site of pUC19
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; recombinant plasmid pJS4M overproducing HsdM compared to HsdS
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EcoR124I HsdR with selenomethionine labeling expressed in Escherichia coli
enzyme with and without a temperature sensitive mutation in the hsdS gene are cloned in pBR322 plamid and introduced into Escherichia coli C3-6
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esp1396I genes expressed in Escherichia coli HB101 and XL1-Blue
synthetic construct
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expressed in Escherichia coli B834 (DE3) cells
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) Gold cells
expressed in Escherichia coli strains BNH670 or GM31 harboring a plasmid with various versions of the EcoRII RM gene complex
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full length HsdR transformed into Escherichia coli B834(DE3) cells
His-tagged gene cloned into the NheI/EcoRI sites of the pTXBI vector and expressed in Escherichia coli ER2566
hsdR PCR product cloned into pMAD, giving rise to pDELTAHsdR-1. Plasmid electroporated into the transformable strain RN4220 at 30C, with erythromycin selection, and subsequently transduced to NCTC8325-4, SH1000 and COL using phage 80alpha
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HsdR subunit cloned into pProExHTc and expressed in Escherichia coli B834(DE3)
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HsdR subunit of isoform EcoR124I, expression in Escherichia coli
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of hsdS(DELTA50)
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PCR-amplified gene for C.PvuII cloned downstream of the arabinose-inducible PBAD promoter in vector pBAD24 yielding plasmids pIM1
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pCR-TOPO/hpyAXII digested with EcoRI and subcloned into plasmid Bluescript SK+. Plasmid pET15b/hpyAXIIR expressed in Escherichia coli strain BL21CodonPlus(DE3)-RIL
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putative HsdM subunit amplified, cloned into pProExHTc, which expresses 25 extra amino acids containing six consecutive His residues at the N-terminus. The expression construct transformed into Escherichia coli B834 (DE3)
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transforming the Escherichia coli BL21(DE3) strains with a BAC C4/1 carrying the hsdR, hsdM and hsdS genes of EcoAO83I and with plasmids carrying the hsdS and hsdM genes of EcoAI
wild-type R.PspGI and its mutant expressed as pET21a-PspGI-WT and pET21a-PspGI-D138A in Escherichia coli strain ER2744
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L80P
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L80P mutation in the modification enzyme of the EcoRII gene complex confers thermosensitivity of cell growth (shows activity at 30C but not at 37C). Under a condition of inhibited protein synthesis, the activity of the mutant is completely lost at a high temperature. In parallel, the L80P mutant protein disappears more rapidly than the wild-type protein
R182A
inactive
T239C
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shows decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme
T402C
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cells carrying the mutation are able to grow at 42C
D354A
activity is significantly impaired
E350A
activity is significantly impaired
F353A
activity is significantly impaired
P349A
activity is significantly impaired
Q344A
similar activity to the wild-type
R351A
activity is negligible
R352A
similar activity to the wild-type
S348A
similar activity to the wild-type
E350A
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activity is significantly impaired
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P349A
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activity is significantly impaired
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Q344A
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similar activity to the wild-type
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R352A
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similar activity to the wild-type
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S348A
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similar activity to the wild-type
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D138A
-
catalytically inactive
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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potential uses for EcoR124I as a nanoactuator within a biosensor in the field of bionanotechnology. It may be used as a molecular dynamo that can measure events from single molecules of DNA, providing a highly sensitive biosensor for detecting events that interrupt translocation events
medicine
additional information
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