EzCatDB: T00055

DB codeT00055
RLCP classification1.15.9400.1180
CATH domainDomain 13.90.241.10
Domain 21.10.10.10
Domain 33.40.91.30Catalytic domain
E.C.3.1.21.4
CSA2fok

CATH domainRelated DB codes (homologues)
1.10.10.10D00510,D00452,D00077,D00517,T00113

Enzyme Name
Swiss-protKEGG

P14870
Protein nameType-2 restriction enzyme FokItype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.FokI
EC 3.1.21.4
Type II restriction enzyme FokI
Type IIS restriction enzyme FokI
Endonuclease FokI


Swiss-prot:Accession NumberP14870
Entry nameT2F1_FLAOK
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
SubunitMonomer. Homodimer when bound to DNA.
Subcellular location
CofactorMagnesium.


CofactorsSubstratesProducts
KEGG-idC00305C00039C00001C00578
CompoundMagnesiumDNAH2ODNA 5'-phosphate
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ion
1fokA01UnboundBound:T-C-G-G-A-T-G-A-T-A-A-C-G-C-T-A-G-T-C-A(chain B)
Unbound
2fokA01UnboundUnbound
Unbound
2fokB01UnboundUnbound
Unbound
1fokA02UnboundUnbound
Unbound
2fokA02UnboundUnbound
Unbound
2fokB02UnboundUnbound
Unbound
1fokA03UnboundUnbound
Unbound
2fokA03UnboundUnbound
Unbound
2fokB03UnboundUnbound
Unbound

Active-site residues
resource
literature [4], [9], [15]
pdbCatalytic residuesCofactor-binding residues
1fokA01

2fokA01

2fokB01

1fokA02

2fokA02

2fokB02

1fokA03LYS 469
ASP 450;ASP 467(two Mg2+ binding)
2fokA03LYS 469
ASP 450;ASP 467(two Mg2+ binding)
2fokB03LYS 469
ASP 450;ASP 467(two Mg2+ binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[15]Fig.2, p.580-582
[20]Fig.1, p.6

references
[1]
PubMed ID2684765
JournalGene
Year1989
Volume80
Pages193-208
AuthorsLooney MC, Moran LS, Jack WE, Feehery GR, Benner JS, Slatko BE, Wilson GG
TitleNucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase.
[2]
PubMed ID1584761
JournalProc Natl Acad Sci U S A
Year1992
Volume89
Pages4275-9
AuthorsLi L, Wu LP, Chandrasegaran S
TitleFunctional domains in Fok I restriction endonuclease.
[3]
PubMed ID8464886
JournalProc Natl Acad Sci U S A
Year1993
Volume90
Pages2764-8
AuthorsLi L, Chandrasegaran S
TitleAlteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis.
[4]
PubMed ID8415747
JournalProc Natl Acad Sci U S A
Year1993
Volume90
Pages9596-600
AuthorsWaugh DS, Sauer RT
TitleSingle amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease.
[5]
PubMed ID7989374
JournalJ Biol Chem
Year1994
Volume269
Pages31978-82
AuthorsKim YG, Li L, Chandrasegaran S
TitleInsertion and deletion mutants of FokI restriction endonuclease.
[6]
PubMed ID7905633
JournalProc Natl Acad Sci U S A
Year1994
Volume91
Pages883-7
AuthorsKim YG, Chandrasegaran S
TitleChimeric restriction endonuclease.
[7]
PubMed ID9042953
JournalFEBS Lett
Year1997
Volume403
Pages136-8
AuthorsHirsch JA, Wah DA, Dorner LF, Schildkraut I, Aggarwal AK
TitleCrystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.
[8]
PubMed ID9426005
JournalGene
Year1997
Volume203
Pages43-9
AuthorsKim YG, Shi Y, Berg JM, Chandrasegaran S
TitleSite-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions.
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.8 ANGSTROMS).
Medline ID97357159
PubMed ID9214510
JournalNature
Year1997
Volume388
Pages97-100
AuthorsWah DA, Hirsch JA, Dorner LF, Schildkraut I, Aggarwal AK
TitleStructure of the multimodular endonuclease FokI bound to DNA.
Related PDB1fok
Related Swiss-protP14870
[10]
PubMed ID9628342
JournalBiol Chem
Year1998
Volume379
Pages489-95
AuthorsKim YG, Smith J, Durgesha M, Chandrasegaran S
TitleChimeric restriction enzyme: Gal4 fusion to FokI cleavage domain.
[11]
PubMed ID9628339
JournalBiol Chem
Year1998
Volume379
Pages467-73
AuthorsStahl F, Wende W, Jeltsch A, Pingoud A
TitleThe mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.
[12]
PubMed ID9519292
JournalCurr Opin Struct Biol
Year1998
Volume8
Pages19-25
AuthorsAggarwal AK, Wah DA
TitleNovel site-specific DNA endonucleases.
[13]
CommentsSUBUNIT.
PubMed ID9724744
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages10570-5
AuthorsBitinaite J, Wah DA, Aggarwal AK, Schildkraut I
TitleFokI dimerization is required for DNA cleavage.
[14]
CommentsX-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Medline ID98393684
PubMed ID9724743
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages10564-9
AuthorsWah DA, Bitinaite J, Schildkraut I, Aggarwal AK
TitleStructure of FokI has implications for DNA cleavage.
Related PDB2fok
Related Swiss-protP14870
[15]
PubMed ID10508668
JournalCurr Opin Chem Biol
Year1999
Volume3
Pages578-83
AuthorsKovall RA, Matthews BW
TitleType II restriction endonucleases: structural, functional and evolutionary relationships.
[16]
PubMed ID9862996
JournalNucleic Acids Res
Year1999
Volume27
Pages674-81
AuthorsSmith J, Berg JM, Chandrasegaran S
TitleA detailed study of the substrate specificity of a chimeric restriction enzyme.
[17]
PubMed ID10715131
JournalBiochemistry
Year2000
Volume39
Pages3097-105
AuthorsHlavaty JJ, Benner JS, Hornstra LJ, Schildkraut I
TitleIdentification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage.
[18]
PubMed ID10654258
JournalJ Mol Evol
Year2000
Volume50
Pages39-44
AuthorsBujnicki JM
TitlePhylogeny of the restriction endonuclease-like superfamily inferred from comparison of protein structures.
[19]
PubMed ID10954606
JournalNucleic Acids Res
Year2000
Volume28
Pages3361-9
AuthorsSmith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D
TitleRequirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.
[20]
PubMed ID10739241
JournalProtein Sci
Year2000
Volume9
Pages1-9
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.
[21]
PubMed ID11491302
JournalJ Mol Biol
Year2001
Volume309
Pages69-78
AuthorsVanamee ES, Santagata S, Aggarwal AK
TitleFokI requires two specific DNA sites for cleavage.
[22]
PubMed ID11113203
JournalMol Cell Biol
Year2001
Volume21
Pages289-97
AuthorsBibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S
TitleStimulation of homologous recombination through targeted cleavage by chimeric nucleases.
[23]
PubMed ID11729187
JournalJ Biol Chem
Year2002
Volume277
Pages4024-33
AuthorsBath AJ, Milsom SE, Gormley NA, Halford SE
TitleMany type IIs restriction endonucleases interact with two recognition sites before cleaving DNA.
[24]
PubMed ID12750473
JournalProc Natl Acad Sci U S A
Year2003
Volume100
Pages6410-5
AuthorsSasnauskas G, Halford SE, Siksnys V
TitleHow the BfiI restriction enzyme uses one active site to cut two DNA strands.
[25]
PubMed ID15247328
JournalNucleic Acids Res
Year2004
Volume32
Pagese95
AuthorsDoi N, Kumadaki S, Oishi Y, Matsumura N, Yanagawa H
TitleIn vitro selection of restriction endonucleases by in vitro compartmentalization.

comments
Although the detailed mechanism of the reaction has not been elucidated, the catalytic site is similar to the other Type II restriction enzymes, such as EcoRV (S00403 in EzCatDB) and BglI (S00405 in EzCatDB). Thus, the catalytic mechanism of this enzyme might be similar to that of those enzymes.
More recent paper [20] supported the substrate-assisted mechanism for the related enzymes (type II restriction enzymes), ruling out the two-metal-ion mechanism. Thus, we concluded that this enzyme adopts the substrate-assisted mechanism with only one metal ion for catalysis (see EcoRV; S00404 in EzCatDB).

createdupdated
2005-08-022009-02-26


Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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