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
3.1.21.5
-
RECOMMENDED NAME
GeneOntology No.
type III site-specific deoxyribonuclease
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
-
-
-
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endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
mechanism
-
endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
mechanism
prophage P1
-
endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
mechanism, formation of loop structures and DNA translocation
-
endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates
show the reaction diagram
mechanism, model of DNA translocation and cleavage, overview
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REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of phosphoric ester
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-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
ATP-dependent type III restriction endonuclease
-
-
BsaHI
Geobacillus stearothermophilus CPW11
B0LX59
-
-
EC 3.1.23
-
-
-
-
EC 3.1.24
-
-
-
-
PspGI
-
recognizes the sequence CCWGG where W is A or T and cuts DNA before the first C in the cognate sequence
PstII
Providencia stuartii 164
-
-
-
R.EcoP15I
-
-
restriction endonuclease PstII
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restriction endonuclease PstII
Providencia stuartii 164
-
-
-
restriction-modification system
-
-
-
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type III DNA restriction/modification enzyme
-
-
type III R/M enzyme
-
-
type III RE
-
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type III restriction endonuclease
-
-
type III restriction enzyme
-
-
-
-
type III restriction enzyme
-
-
type III RM system
Q5F957
-
type III testriction-modification enzyme
-
-
type III-like restriction endonuclease
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-
additional information
-
a complete listing of all these enzymes and their recognition sites has been produced by R.J. Roberts, this list is updated annually
CAS REGISTRY NUMBER
COMMENTARY
9075-08-5
not distinguished from EC 3.1.21.4
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain ATCC 10987, enzyme BceS1
SwissProt
Manually annotated by BRENDA team
EcoP15; EcoPI
-
-
Manually annotated by BRENDA team
EcoPI; enzyme EcoP15I
-
-
Manually annotated by BRENDA team
enzyme EcoK
-
-
Manually annotated by BRENDA team
enzyme EcoP1I
-
-
Manually annotated by BRENDA team
enzymes EcoP1I, EcoP15I
-
-
Manually annotated by BRENDA team
isoform EcoP15I
-
-
Manually annotated by BRENDA team
mutant enzymes: R.EcoPIK90R, R.EcoPIT91A and R.EcoPIH229K; R.EcoPI
-
-
Manually annotated by BRENDA team
Escherichia coli P15I
P15I
-
-
Manually annotated by BRENDA team
enzyme HinFIII
-
-
Manually annotated by BRENDA team
type III restriction-modification system
-
-
Manually annotated by BRENDA team
enzyme LlaFI
-
-
Manually annotated by BRENDA team
resNgoAXP restriction modification protein
UniProt
Manually annotated by BRENDA team
prophage P1
enzyme EcoP1I
-
-
Manually annotated by BRENDA team
; strain 164
-
-
Manually annotated by BRENDA team
Providencia stuartii 164
strain 164
-
-
Manually annotated by BRENDA team
strain GI-H
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-
Manually annotated by BRENDA team
different strains
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-
Manually annotated by BRENDA team
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
DNA + H2O
specific double-stranded DNA fragments with terminal 5'-phosphate
show the reaction diagram
-
action of type III restriction enzymes takes place on replicated or replicating DNA in VIVO and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombinantion reconstitutes an intact DNA from them, and that REcBCD exonuclease blocks this repair by degradation from the restriction breaks
-
-
?
DNA + H2O
specific double-stranded DNA fragments with terminal 5'-phosphate
show the reaction diagram
Providencia stuartii, Providencia stuartii 164
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the intact Res2Mod2 tetramer is a fast endonuclease and slow methyltransferase, thereby favouring DNA cleavage, subassemblies of PstII in which the Res subunits have dissociated are more efficient methyltransferases. DNA cleavage by these lower molecular weight species may only occur if sufficient hsdR associates to form a Res2Mod2 tetramer before methylation occurs. This dynamic association of Res and Mod might play a key role in the control of PstII activity in vivo
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-
?
additional information
?
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-
the enzyme uses both diffuse DNA loop formation and ATPase driven translocation of the intervening DNA contour to communicate between two recognition sites
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-
-
additional information
?
-
-
type III restriction-modification systems play only a minor role in the overall defence of the cell against invasion by foreign DNA
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-
-
additional information
?
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on linear DNA in contrast, there is only one route and resolvase completely blocks communication and cleavage, while on circular DNA, there are two routes for communication. If one route is blocked by resolvase, the enzyme can use the alternative route. No stepwise motor mechanism for type III enzymes, the enzyme can bypass triplexes during sliding
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-
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INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
HU protein
-
binding of HU protein interfers with R.EcoP15I cleavage activity
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Lac repressor
-
cleavage can be abolished by the binding of Lac repressor downstream (3' side) but not upstream (5' side) of the recognition site
-
additional information
-
type III restriction is alleviated by bacteriophage (RecE) homologous recombination function
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additional information
-
sinefungin neither has an appreciable effect on DNA cleavage by EcoP15I nor compensated for the second recognition site; the catalytic activity of EcoP15I after 3 h of pre-incubation without DNA is strongly decreased
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pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
80
-
-
the optimal temperature for DNA cleavage by PspGI is at 80°C or above
PDB
SCOP
CATH
ORGANISM
Bacillus subtilis (strain 168)
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
tetramer
Escherichia coli, prophage P1
-
2 * 105000 + 2 * 74000, deduced from gene sequence
tetramer
-
the intact Res2Mod2 tetramer is a fast endonuclease and slow methyltransferase, thereby favouring DNA cleavage, subassemblies of PstII in which the Res subunits have dissociated are more efficient methyltransferases. DNA cleavage by these lower molecular weight species may only occur if sufficient hsdR associates to form a Res2Mod2 tetramer before methylation occurs. This dynamic association of Res and Mod might play a key role in the control of PstII activity in vivo
tetramer
Providencia stuartii 164
-
the intact Res2Mod2 tetramer is a fast endonuclease and slow methyltransferase, thereby favouring DNA cleavage, subassemblies of PstII in which the Res subunits have dissociated are more efficient methyltransferases. DNA cleavage by these lower molecular weight species may only occur if sufficient hsdR associates to form a Res2Mod2 tetramer before methylation occurs. This dynamic association of Res and Mod might play a key role in the control of PstII activity in vivo
-
?
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x * 106000, subunit Res, + x * 75000, subunit Mod, deduced from gene sequence
additional information
-
separation of Res and Mod subunits by gel filtration in presence of 2 M NaCl
additional information
-
functional interaction of enzymes EcoP1I and EcoP15I
additional information
-
in the presence of specific DNA, the entire modification subunit of the enzyme is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the modification subunit are not detected. In contrast, the restriction subunit is comprised of two trypsin-resistant domains of about 77000-79000 Da and 27000-29000 Da, respectively. Both structural restriction domains are connected by a flexible linker region that spans 23 amino acid residues
additional information
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the type III R/M enzymes comprise two modification subunits, each containing a target recognition domain to bind to the target sequence and a methyltransferase catalytic domain to monitor the methylation status of an adenine in the target, and two restriction subunits each containing a DNA helicase and ATP-hydrolysing domain and an endonuclease DNA cleavage domain
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
95
-
-
half life of 2 h at 95°C
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
EcoP15I is stabilized in the presence of specific DNA
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Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Ni-NTA agarose column chromatography
B0LX59
P11 cellulose phosphate column chromatography
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D898A
-
Res subunit, no enzymic activity
E916A
-
Res subunit, no enzymic activity
G448S
-
mutant of Mod subunit, not able to bind S-adenosyl-L-methionine, no DNA cleavage
R534A
-
Res subunit, no enzymic activity
D354A
B0LX59
mutant shows reduced activity compared to the wild type enzyme
E350A
B0LX59
mutant shows reduced activity compared to the wild type enzyme
F353A
B0LX59
mutant shows reduced activity compared to the wild type enzyme
P349A
B0LX59
mutant shows reduced activity compared to the wild type enzyme
Q344A
B0LX59
mutant shows activity similar to wild type enzyme
R351A
B0LX59
the activity of the R351A mutant is negligible
R352A
B0LX59
mutant shows activity similar to the wild type enzyme
D898A
prophage P1
-
Res subunit, no enzymic activity
E916A
prophage P1
-
Res subunit, no enzymic activity
K918A
prophage P1
-
Res subunit, no enzymic activity
P897A
prophage P1
-
Res subunit, activity similar to wild type
R534A
prophage P1
-
Res subunit, no enzymic activity
D138A
-
catalytically inactive
K918A
-
Res subunit, no enzymic activity
additional information
-
mutant c2-134, unable to cleave DNA, mutant c2-440, poor ability to cleave DNA
additional information
-
study on the predicted linker region between the two domains of the restriction subunit by insertional mutagenesis. Introduction of up to 18 amino acids in the N- and C-terminal region of the linker. The region tolerated the introduced genetic alterations without loss of catalytic function or changes in cleavage position
S348A
B0LX59
mutant shows activity similar to the wild type enzyme
additional information
-
analysis of several strains for phase variable methyltransferase activity, mod, and restriction endonuclease activity. Of 41 strains in the survey that contain a phase variable mod gene, seven have an obvious mutation in the restriction domain and appear to be dedicated phasevarions. The remaining 15 strains that do not contain a phase variable mod gene are likely to be dedicated, functional, type III restriction-modification systems
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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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