FokI
| Restriction endonuclease FokI, C terminal | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| File:1fok.png | |||||||||
| Identifiers | |||||||||
| Symbol | Endonuc-FokI_C | ||||||||
| Pfam | PF09254 | ||||||||
| Pfam clan | CL0415 | ||||||||
| InterPro | IPR015334 | ||||||||
| SCOP | 2fok | ||||||||
| SUPERFAMILY | 2fok | ||||||||
|
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The enzyme FokI, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal.[2] Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.[3]
Its molecular mass is 65.4 kDa, being composed of 587 amino acids.
Contents
DNA-binding domain
The recognition domain contains three subdomains (D1, D2 and D3) that are evolutionarily related to the DNA-binding domain of the catabolite gene activator protein which contains a helix-turn-helix.[3]
DNA-cleavage domain
DNA cleavage is mediated through the non-specific cleavage domain which also includes the dimerisation surface.[4] The dimer interface is formed by the parallel helices α4 and α5 and two loops P1 and P2 of the cleavage domain.[3]
Activity
When the nuclease is unbound to DNA, the endonuclease domain is sequestered by the DNA-binding domain and is released through a conformational change in the DNA-binding domain upon binding to its recognition site. Cleavage only occurs upon dimerisation, when the recognition domain is bound to its cognate site and in the presence of magnesium ions.[4]
Exploitation
The endonuclease domain of FokI has been used in several studies, after combination with a variety of DNA-binding domains such as the zinc finger (see zinc finger nuclease),[2] or inactive Cas9[5][6][7]
One of several human vitamin D receptor gene variants is a single nucleotide polymorphism in the start codon of the gene which can be distinguished through the use of the FokI enzyme.[8]
References
- ↑ Lua error in package.lua at line 80: module 'strict' not found.
- ↑ 2.0 2.1 Lua error in package.lua at line 80: module 'strict' not found.
- ↑ 3.0 3.1 3.2 Lua error in package.lua at line 80: module 'strict' not found.
- ↑ 4.0 4.1 Lua error in package.lua at line 80: module 'strict' not found.
- ↑ Tsai, S. Q. et al. (2014). Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 doi:10.1038/nbt.2908
- ↑ Guilinger, J. P., Thompson, D. B. & Liu, D. R. (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature Biotechnol. 32, 577–582 doi:10.1038/nbt.2909
- ↑ Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K. & Tsai, S. Q. (2015). Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 doi:10.1089/hum.2015.084
- ↑ Lua error in package.lua at line 80: module 'strict' not found.
