Information on EC 3.1.21.5 - type III site-specific deoxyribonuclease

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

EC NUMBER
COMMENTARY hide
3.1.21.5
-
RECOMMENDED NAME
GeneOntology No.
type III site-specific deoxyribonuclease
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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-
-
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CAS REGISTRY NUMBER
COMMENTARY hide
9075-08-5
not distinguished from EC 3.1.21.4
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
P15I
-
-
Manually annotated by BRENDA team
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C7LKU0
UniProt
Manually annotated by BRENDA team
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C7LKU0
UniProt
Manually annotated by BRENDA team
prophage P1
enzyme EcoP1I
-
-
Manually annotated by BRENDA team
strain 164
-
-
Manually annotated by BRENDA team
strain GI-H
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-
Manually annotated by BRENDA team
different strains
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
DNA + H2O
specific double-stranded DNA fragments with terminal 5'-phosphate
show the reaction diagram
phage lambda DNA + H2O
?
show the reaction diagram
restriction activity requires the presence of both the restriction modification endonuclease protein and the methylase protein of the NgoAXP restriction modification system
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-
-
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
specific double-stranded DNA fragments with terminal 5'-phosphate
show the reaction diagram
additional information
?
-
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
EDTA
C7LKU0
complete inhibition at 0.02 mM
HU protein
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binding of HU protein interfers with R.EcoP15I cleavage activity
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Lac repressor
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cleavage can be abolished by the binding of Lac repressor downstream (3' side) but not upstream (5' side) of the recognition site
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additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid
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may substitute for S-adenosyl-L-methionine
S-adenosylmethionine
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stimulates
S6-methyl-6-deaminosinefungin
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may substitute for S-adenosyl-L-methionine
sinefungin
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may substitute for S-adenosyl-L-methionine
additional information
-
type III restriction is enhanced by bacterial (RecBCD) function
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
80
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the optimal temperature for DNA cleavage by PspGI is at 80°C or above
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
235700
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calculated from amino acid sequence
258400
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EcoPI, calculated from amino acid sequence
259100
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EcoP15I, calculated from amino acid sequence
259300
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EcoP15I, multi-angle light scattering
407000
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sedimentation studies
409000
prophage P1
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sedimentation studies
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 106000, subunit Res, + x * 75000, subunit Mod, deduced from gene sequence
heterotrimer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
database information: http://rebase.neb.com
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real-time imaging of enzyme at scan rates of 1-3 frames per s. EcoP15I translocates DNA in an ATP-dependent manner, at a rate of 79 bp/s, resulting in the accumulation of supercoiling. EcoP15I bound to its recognition site also makes nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites
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study of enzyme-DNA pre-cleavage complexes by atomic force microscopy. DNA loops observed are formed by a contact between site-bound EcoP15I enzyme and a nonspecific region of DNA, and do not result from translocation. Model for restricition by type III enzymes involving both structural elements, and a functional reason for the unusual site orientation required for the cleavage reaction
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
95
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half life of 2 h at 95°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
EcoP15I is stabilized in the presence of specific DNA
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose column chromatography
P11 cellulose phosphate column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6His-tag
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expressed in Escherichia coli strain ER2566
expressed in Escherichia coli strain ER2744
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expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D898A
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Res subunit, no enzymic activity
E916A
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Res subunit, no enzymic activity
G448S
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mutant of Mod subunit, not able to bind S-adenosyl-L-methionine, no DNA cleavage
K918A
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Res subunit, no enzymic activity
R534A
-
Res subunit, no enzymic activity
D354A
mutant shows reduced activity compared to the wild type enzyme
E350A
mutant shows reduced activity compared to the wild type enzyme
F353A
mutant shows reduced activity compared to the wild type enzyme
P349A
mutant shows reduced activity compared to the wild type enzyme
Q344A
mutant shows activity similar to wild type enzyme
R351A
the activity of the R351A mutant is negligible
R352A
mutant shows activity similar to the wild type enzyme
S348A
mutant shows activity similar to the wild type enzyme
D898A
prophage P1
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Res subunit, no enzymic activity
E916A
prophage P1
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Res subunit, no enzymic activity
K918A
prophage P1
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Res subunit, no enzymic activity
P897A
prophage P1
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Res subunit, activity similar to wild type
R534A
prophage P1
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Res subunit, no enzymic activity
D138A
-
catalytically inactive
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reconstitution of separated Res and Mod subunits leads to an inactive apoenzyme lacking S-adenosyl-L-methionine, reconstitution in presence of S-adenosyl L-methionine was not successful
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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transcriptome analysis
additional information
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inactivation of this restriction system dramatically increases the transformation efficiency of clinical Staphylococcus aureus methicillin-resistant strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin
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