Information on EC 3.1.21.4 - type II site-specific deoxyribonuclease and Organism(s) Escherichia coli and UniProt Accession P00642

Go to AA Sequence and Transmembrane Helices SearchGo to Localization Prediction

P00642 pBLAST

T2E1_ECOLX

277

31059

Swiss-Prot

Show Sequence

Go to PDB and Structure Links Search

2 entries

Go to Molecular Weight Search

26000

2 * 26000, calculated

28500

enzyme EcoRI

30000

x * 30000, EcoRI, SDS-PAGE

730520

54000 - 57000

gel-filtration, wild-type and mutants

62000

gel filtration

67000

calculated molecular mass of Eco31I

dimer

homodimer

additional information

the isolated C-terminal domain dimer has an interface that binds a single cognate DNA molecule whereas the N-terminal domain is a monomer that also binds a single copy of cognate DNA

Go to Crystallization (commentary) Search

crystal structure of EcoO109I and its complex with DNA

Go to Protein Variants Search

665694

database information: http://rebase.neb.com

mutant R88A

D299A

no catalytic activity, mutant like wild-type remains in dimeric form

D329A

less than 0.01% residual activity, mutant like wild-type remains in dimeric form

E271A

no catalytic activity, mutant like wild-type remains in dimeric form

E337A

less than 0.01% residual activity, mutant like wild-type remains in dimeric form

K263A

inactive mutant with truncated EcoRII N-domain

693058

K324A

no catalytic activity, mutant like wild-type remains in dimeric form

K328A

less than 0.01% residual activity, mutant like wild-type remains in dimeric form

L80P

P14633

the mutant enzyme shows decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the wild type enzyme, the activity of the L80P mutant is completely lost at a high temperature

692848

M357P

mutation converts DNA methyltransferase to a type II endonuclease with 5'-CAGCAG-3' restriction site that cleaves only supercoiled DNA but does not act on nicked or linearized DNA

683660

R330A

2 entries

R88A

crystallization data, autoinhibition/activation mechanism

652931

Go to pH Stability Search

6 - 10

stable from pH 6.0 to pH 10.0

663677

Go to Temperature Stability Search

30 - 42

Go to Purification (commentary) Search

P14633

the extract from cells expressing the wild type EcoRII shows the activity both at 30°C and 37°C, the extract from cells expressing the L80P mutant form shows activity at 30°C but not at 37°C, the L80P mutant protein is significantly unstable at 42°C compared with the wild type protein

stable up to

ammonium sulfate fractionation, phosphocellulose P-11 column chromatography, heparin-Sepharose column chromatography, AH-Sepharose column chromatography, and Blue-Sepharose column chromatography

EcoRI and EcoRV

extremely fast and economical method of restriction endonucleases free from contaminating nuclease activity by a combination of affity partitioning and ion-exchange chromatography

134264

HiTrap heparin column chromatography

partial

phosphocellulose P-11 column chromatography, heparin Sepharose column chromatography, CM Sephadex C-50 gel filtration or Blue-agarose column chromatography

728861

Go to Cloned (commentary) Search

cloning of the complete restriction-modification system in Escherichia coli

134265

expressed in Escherichia coli strain BNH670

P14633

692848

expressed in Escherichia coli strain JM109

693058

expressed in Escherichia coli strains ER2267 and HMS174

Go to Application Search

analysis

method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels via linear dichroism spectroscopy

683156

molecular biology

top print hide References Search

P25239, Q5ZND2

a straightforward, general and automatable model system for studying the activity of restriction endonucleases by using massively parallel sequencing is described, which should be highly applicable for the future studies of large sets of restriction endonucleases and their activity

751913

134251

Roberts, R.J.

Restriction enzymes and their isoschizomers

Nucleic Acids Res.

2331-2365

1990

Acetobacter aceti, Gluconobacter oxydans, Acetobacter pasteurianus, Acinetobacter lwoffii, Streptomyces phaeochromogenes, Thalassobius gelatinovorus, Anabaena cylindrica, Dolichospermum flos-aquae, Anabaena subcylindrica, Trichormus variabilis, Synechocystis sp., Aphanothece halophytica, Cellulosimicrobium cellulans, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Brevibacillus brevis, [Bacillus] caldolyticus, Bacillus cereus, Weizmannia coagulans, Bacillus subtilis, Bacillus pumilus, Lysinibacillus sphaericus, uncultured bacterium, Bifidobacterium longum subsp. infantis, Curtobacterium albidum, Caryophanon latum, Chloroflexus aurantiacus, Chromobacterium violaceum, Citrobacter freundii, Dactylococcus salina, Deinococcus radiodurans, Deinococcus radiophilus, Desulfovibrio desulfuricans, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecalis, Eucapsis sp., Flavobacterium aquatile, Chryseobacterium indologenes, Planomicrobium okeanokoites, Thermus thermophilus, Frankia sp., Fusobacterium nucleatum, Haemophilus aegyptius, Avibacterium paragallinarum, Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Herpetosiphon aurantiacus, Klebsiella pneumoniae, Methanothermobacter wolfeii, Methanococcus aeolicus, Methylophilus methylotrophus, Psychrobacter urativorans, Micrococcus luteus, Micrococcus lylae, Staphylococcus aureus, Microcoleus sp., Moraxella bovis, Moraxella nonliquefaciens, Mycoplasmopsis fermentans, Bergeriella denitrificans, Neisseria lactamica, Neisseria mucosa heidelbergensis, Lentzea aerocolonigenes, Nocardia argentinensis, Gordonia rubripertincta, Nocardia otitidiscaviarum, Rhodococcus ruber, Nostoc sp., Plesiomonas shigelloides, Proteus vulgaris, Pseudomonas sp., Pseudomonas putida, Pseudomonas fluorescens, Paucimonas lemoignei, Stenotrophomonas maltophilia, Pseudomonas stutzeri, Rhizobium leguminosarum, Cereibacter sphaeroides, Salmonella enterica subsp. enterica serovar Typhi, Serratia marcescens, Sphaerotilus natans, Sphaerotilus sp., Arthrospira platensis, Streptomyces achromogenes, Lactococcus cremoris, Enterococcus durans, Streptomyces albus, Streptomyces caespitosus, Streptomyces fimbriatus, Thermus aquaticus, Thermus filiformis, Thermus sp., Vibrio sp., Xanthomonas campestris pv. badrii, Xanthomonas campestris, Xanthomonas phaseoli pv. manihotis, Xanthomonas vasicola, Xanthomonas citri pv. malvacearum, Acetobacter pasteurianus ApaLI

2159140

134252

Roberts, R.J.

Restriction and modification enzymes and their recognition sequences

Nucleic Acids Res.

r135-r167

1983

Escherichia coli

6306557

134254

Roberts, R.J.; Macelis, D.

REBASE - restriction enzymes and methylases

Nucleic Acids Res.

268-269

2001

Escherichia coli

11125108

134259

Kruger, D.H.; Kupper, D.; Meisel, A.; Tierlich, M.; Reuter, M.; Schroeder, C.

Restriction endonucleases functionally interacting with two DNA sites

Gene

157

165

1995

Escherichia coli

7607484

134262

Wells, R.D.; Klein, R.D.; Singleton, C.K.

Type II restriction enzymes

The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )

157-191

1981

Trichormus variabilis, Cellulosimicrobium cellulans, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, [Bacillus] caldolyticus, Bacillus subtilis, Lysinibacillus sphaericus, Desulfovibrio desulfuricans, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecalis, Thermus thermophilus, Fusobacterium nucleatum, Haemophilus aegyptius, Avibacterium paragallinarum, Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Herpetosiphon aurantiacus, Staphylococcus aureus, Moraxella bovis, Providencia stuartii, Cereibacter sphaeroides, Serratia marcescens, Streptomyces achromogenes, Streptomyces albus, Xanthomonas campestris pv. badrii, Xanthomonas vasicola, Xanthomonas citri pv. malvacearum, Staphylococcus aureus Sau96I

134263

Molloy, P.L.; Symons, R.H.

Cleavage of DNA.RNA hybrids by type II restriction enzymes

Nucleic Acids Res.

2939-2946

1980

Cellulosimicrobium cellulans, Bacillus amyloliquefaciens, Escherichia coli, Haemophilus aegyptius, Haemophilus haemolyticus, Thermus aquaticus

6253897

134264

Vlatakis, G.; Bouriotis, V.

Affinity partitioning of restriction endonucleases. Application to the purification of EcoR I and EcoR V

J. Chromatogr.

538

311-321

1991

Escherichia coli

2016384

134265

Wilson, G.G.

Type II restriction-modification systems

Trends Genet.

314-318

1988

Dolichospermum flos-aquae, Trichormus variabilis, Bacillus amyloliquefaciens, Bacillus subtilis, Desulfovibrio desulfuricans, Streptococcus pneumoniae, Escherichia coli, Planomicrobium okeanokoites, Haemophilus aegyptius, Haemophilus haemolyticus, Haemophilus influenzae, Herpetosiphon aurantiacus, Methanothermobacter wolfeii, Proteus vulgaris, Providencia stuartii, Streptomyces albus, Thermus aquaticus, Xanthomonas campestris pv. badrii

3070854

134268

Glenn, T.C.; Waller, D.R.; Braun, M.J.

Increasing proportions of uracil in DNA substrates increases inhibition of restriction enzyme digests

Biotechniques

1086-1090

1994

Acetobacter pasteurianus, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Caryophanon latum, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Nocardia otitidiscaviarum, Pseudomonas sp., Serratia marcescens, Streptomyces achromogenes, Xanthomonas campestris pv. badrii, Acetobacter pasteurianus ApaI, Geobacillus stearothermophilus BstXI, Geobacillus stearothermophilus BstZI

7873177

649233

Zhou, X.E.; Wang, Y.; Reuter, M.; Mackeldanz, P.; Kruger, D.H.; Meehan, E.J.; Chen, L.

A single mutation of restriction endonuclease EcoRII led to a new crystal form that diffracts to 2.1 A resolution

Acta Crystallogr. Sect. D

910-912

2003

Escherichia coli

12777809

652931

Zhou, X.E.; Wang, Y.; Reuter, M.; Mucke, M.; Kruger, D.H.; Meehan, E.J.; Chen, L.

Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold

J. Mol. Biol.

335

307-319

2004

Escherichia coli

14659759

663677

Lazim, H.; Josephsen, J.; Ben Hassen, A.; Belhadj, O.; Limam, F.

Eco1524I, a type II restriction endonuclease: isolation, partial purification, and characterization

Appl. Biochem. Biotechnol.

125

189-199

2005

Escherichia coli

15917582

664054

Baskunov, V.B.; Subach, F.V.; Kolbanovskiy, A.; Kolbanovskiy, M.; Eremin, S.A.; Johnson, F.; Bonala, R.; Geacintov, N.E.; Gromova, E.S.

Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease

Biochemistry

1054-1066

2005

Escherichia coli

15654762

665694

Hashimoto, H.; Shimizu, T.; Imasaki, T.; Kato, M.; Shichijo, N.; Kita, K.; Sato, M.

Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA

J. Biol. Chem.

280

5605-5610

2005

Escherichia coli

15590682

666080

Tamulaitis, G.; Sasnauskas, G.; Mucke, M.; Siksnys, V.

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

J. Mol. Biol.

358

406-419

2006

Escherichia coli

16529772

666493

van den Broek, B.; Noom, M.C.; Wuite, G.J.

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Nucleic Acids Res.

2676-2684

2005

Bacillus amyloliquefaciens, Escherichia coli

15886396

666494

Nikolajewa, S.; Beyer, A.; Friedel, M.; Hollunder, J.; Wilhelm, T.

Common patterns in type II restriction enzyme binding sites

Nucleic Acids Res.

2726-2733

2005

Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Citrobacter freundii, Escherichia coli, Planomicrobium okeanokoites, Haemophilus influenzae, Moraxella sp., Mycoplasma sp., Neisseria gonorrhoeae, Lentzea aerocolonigenes, Proteus vulgaris

15888729

683067

Takahashi, S.; Matsuno, H.; Furusawa, H.; Okahata, Y.

Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance

Anal. Biochem.

361

210-217

2007

Escherichia coli

17217909

683156

Hicks, M.R.; Rodger, A.; Thomas, C.M.; Batt, S.M.; Dafforn, T.R.

Restriction enzyme kinetics monitored by UV linear dichroism

Biochemistry

8912-8917

2006

Escherichia coli, Klebsiella pneumoniae, Bergeriella denitrificans, Neisseria lactamica, Nocardia otitidiscaviarum, Xanthomonas campestris pv. badrii

16846234