DB code: T00055

RLCP classification 1.15.9400.1180 : Hydrolysis
CATH domain 3.90.241.10 : FokI Restriction Endonuclease; Chain A, domain 1
1.10.10.10 : Arc Repressor Mutant, subunit A
3.40.91.30 : Restriction Endonuclease Catalytic domain
E.C. 3.1.21.4
CSA 2fok
M-CSA 2fok
MACiE

CATH domain Related DB codes (homologues)
1.10.10.10 : Arc Repressor Mutant, subunit A D00510 D00452 D00077 D00517 T00113

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P14870 Type-2 restriction enzyme FokI
R.FokI
EC 3.1.21.4
Type II restriction enzyme FokI
Type IIS restriction enzyme FokI
Endonuclease FokI
PF09254 (Endonuc-FokI_C)
PF02980 (FokI_C)
PF02981 (FokI_N)
[Graphical View]

KEGG enzyme name
type II site-specific deoxyribonuclease
type II restriction enzyme

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P14870 T2F1_FLAOK Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates. Monomer. Homodimer when bound to DNA. Magnesium.

KEGG Pathways
Map code Pathways E.C.

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00039 C00001 C00578
E.C.
Compound Magnesium DNA H2O DNA 5'-phosphate
Type divalent metal (Ca2+, Mg2+) nucleic acids H2O nucleic acids,phosphate group/phosphate ion
ChEBI 18420
 18420
 		15377
 15377
PubChem 888
 888
 		22247451
 962
 22247451
 962
1fokA01 Unbound Bound:T-C-G-G-A-T-G-A-T-A-A-C-G-C-T-A-G-T-C-A (chain B) Unbound
2fokA01 Unbound Unbound Unbound
2fokB01 Unbound Unbound Unbound
1fokA02 Unbound Unbound Unbound
2fokA02 Unbound Unbound Unbound
2fokB02 Unbound Unbound Unbound
1fokA03 Unbound Unbound Unbound
2fokA03 Unbound Unbound Unbound
2fokB03 Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [4], [9], [15]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1fokA01
2fokA01
2fokB01
1fokA02
2fokA02
2fokB02
1fokA03 LYS 469 ASP 450;ASP 467(two Mg2+ binding)
2fokA03 LYS 469 ASP 450;ASP 467(two Mg2+ binding)
2fokB03 LYS 469 ASP 450;ASP 467(two Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[15]
Fig.2, p.580-582
[20]
Fig.1, p.6

References
[1]
Resource
Comments
Medline ID
PubMed ID 2684765
Journal Gene
Year 1989
Volume 80
Pages 193-208
Authors Looney MC, Moran LS, Jack WE, Feehery GR, Benner JS, Slatko BE, Wilson GG
Title Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 1584761
Journal Proc Natl Acad Sci U S A
Year 1992
Volume 89
Pages 4275-9
Authors Li L, Wu LP, Chandrasegaran S
Title Functional domains in Fok I restriction endonuclease.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 8464886
Journal Proc Natl Acad Sci U S A
Year 1993
Volume 90
Pages 2764-8
Authors Li L, Chandrasegaran S
Title Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 8415747
Journal Proc Natl Acad Sci U S A
Year 1993
Volume 90
Pages 9596-600
Authors Waugh DS, Sauer RT
Title Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 7989374
Journal J Biol Chem
Year 1994
Volume 269
Pages 31978-82
Authors Kim YG, Li L, Chandrasegaran S
Title Insertion and deletion mutants of FokI restriction endonuclease.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 7905633
Journal Proc Natl Acad Sci U S A
Year 1994
Volume 91
Pages 883-7
Authors Kim YG, Chandrasegaran S
Title Chimeric restriction endonuclease.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 9042953
Journal FEBS Lett
Year 1997
Volume 403
Pages 136-8
Authors Hirsch JA, Wah DA, Dorner LF, Schildkraut I, Aggarwal AK
Title Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 9426005
Journal Gene
Year 1997
Volume 203
Pages 43-9
Authors Kim YG, Shi Y, Berg JM, Chandrasegaran S
Title Site-specific cleavage of DNA-RNA hybrids by zinc finger/FokI cleavage domain fusions.
Related PDB
Related UniProtKB
[9]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.8 ANGSTROMS).
Medline ID 97357159
PubMed ID 9214510
Journal Nature
Year 1997
Volume 388
Pages 97-100
Authors Wah DA, Hirsch JA, Dorner LF, Schildkraut I, Aggarwal AK
Title Structure of the multimodular endonuclease FokI bound to DNA.
Related PDB 1fok
Related UniProtKB P14870
[10]
Resource
Comments
Medline ID
PubMed ID 9628342
Journal Biol Chem
Year 1998
Volume 379
Pages 489-95
Authors Kim YG, Smith J, Durgesha M, Chandrasegaran S
Title Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 9628339
Journal Biol Chem
Year 1998
Volume 379
Pages 467-73
Authors Stahl F, Wende W, Jeltsch A, Pingoud A
Title The 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.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 9519292
Journal Curr Opin Struct Biol
Year 1998
Volume 8
Pages 19-25
Authors Aggarwal AK, Wah DA
Title Novel site-specific DNA endonucleases.
Related PDB
Related UniProtKB
[13]
Resource
Comments SUBUNIT.
Medline ID
PubMed ID 9724744
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 10570-5
Authors Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I
Title FokI dimerization is required for DNA cleavage.
Related PDB
Related UniProtKB
[14]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Medline ID 98393684
PubMed ID 9724743
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 10564-9
Authors Wah DA, Bitinaite J, Schildkraut I, Aggarwal AK
Title Structure of FokI has implications for DNA cleavage.
Related PDB 2fok
Related UniProtKB P14870
[15]
Resource
Comments
Medline ID
PubMed ID 10508668
Journal Curr Opin Chem Biol
Year 1999
Volume 3
Pages 578-83
Authors Kovall RA, Matthews BW
Title Type II restriction endonucleases: structural, functional and evolutionary relationships.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 9862996
Journal Nucleic Acids Res
Year 1999
Volume 27
Pages 674-81
Authors Smith J, Berg JM, Chandrasegaran S
Title A detailed study of the substrate specificity of a chimeric restriction enzyme.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 10715131
Journal Biochemistry
Year 2000
Volume 39
Pages 3097-105
Authors Hlavaty JJ, Benner JS, Hornstra LJ, Schildkraut I
Title Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID 10654258
Journal J Mol Evol
Year 2000
Volume 50
Pages 39-44
Authors Bujnicki JM
Title Phylogeny of the restriction endonuclease-like superfamily inferred from comparison of protein structures.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID 10954606
Journal Nucleic Acids Res
Year 2000
Volume 28
Pages 3361-9
Authors Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D
Title Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.
Related PDB
Related UniProtKB
[20]
Resource
Comments
Medline ID
PubMed ID 10739241
Journal Protein Sci
Year 2000
Volume 9
Pages 1-9
Authors Dall'Acqua W, Carter P
Title Substrate-assisted catalysis: molecular basis and biological significance.
Related PDB
Related UniProtKB
[21]
Resource
Comments
Medline ID
PubMed ID 11491302
Journal J Mol Biol
Year 2001
Volume 309
Pages 69-78
Authors Vanamee ES, Santagata S, Aggarwal AK
Title FokI requires two specific DNA sites for cleavage.
Related PDB
Related UniProtKB
[22]
Resource
Comments
Medline ID
PubMed ID 11113203
Journal Mol Cell Biol
Year 2001
Volume 21
Pages 289-97
Authors Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S
Title Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.
Related PDB
Related UniProtKB
[23]
Resource
Comments
Medline ID
PubMed ID 11729187
Journal J Biol Chem
Year 2002
Volume 277
Pages 4024-33
Authors Bath AJ, Milsom SE, Gormley NA, Halford SE
Title Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA.
Related PDB
Related UniProtKB
[24]
Resource
Comments
Medline ID
PubMed ID 12750473
Journal Proc Natl Acad Sci U S A
Year 2003
Volume 100
Pages 6410-5
Authors Sasnauskas G, Halford SE, Siksnys V
Title How the BfiI restriction enzyme uses one active site to cut two DNA strands.
Related PDB
Related UniProtKB
[25]
Resource
Comments
Medline ID
PubMed ID 15247328
Journal Nucleic Acids Res
Year 2004
Volume 32
Pages e95
Authors Doi N, Kumadaki S, Oishi Y, Matsumura N, Yanagawa H
Title In vitro selection of restriction endonucleases by in vitro compartmentalization.
Related PDB
Related UniProtKB

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).

Created Updated
2005-08-02 2009-02-26