FokI explained

Symbol:	Endonuc-Fok1_C
Restriction endonuclease Fok1, C terminal
Pfam:	PF09254
Pfam Clan:	CL0415
Interpro:	IPR015334
Scop:	2fok

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non sequence-specific DNA cleavage domain at the C-terminal.^[1] 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 the DNA at two locations, regardless of the nucleotide sequence at the cut site. The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand,^[2] producing two sticky ends with 4-bp overhangs.

Its molecular mass is 65.4 kDa, being composed of 587 amino acids.

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.^[2]

DNA-cleavage domain

DNA cleavage is mediated through the non-specific cleavage domain which also includes the dimerisation surface.^[3] The dimer interface is formed by the parallel helices α4 and α5 and two loops P1 and P2 of the cleavage domain.^[2]

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 dimerization, when the recognition domain is bound to its cognate site and in the presence of magnesium ions.^[3]

Exploitation

The endonuclease domain of Fok1 has been used in several studies, after combination with a variety of DNA-binding domains such as the zinc finger (see zinc finger nuclease),^[1] or inactive Cas9^[4] ^[5] ^[6]

One of several human vitamin D receptor gene variants is caused by a single nucleotide polymorphism in the start codon of the gene which can be distinguished through the use of the Fok1 enzyme.^[7]

References

Durai S, Mani M, Kandavelou K, Wu J, Porteus M, Chandrasegaran S . Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells . Nucleic Acids Res . 33 . 18 . 5978–90 . 2005 . 16251401 . 10.1093/nar/gki912 . 1270952.
Wah . D. A. . Bitinaite, J. . Schildkraut, I. . Aggarwal, A. K. . 1998 . Structure of Fok1 has implications for DNA cleavage . Proc Natl Acad Sci USA . 95 . 18 . 10564–9 . 10.1073/pnas.95.18.10564 . 9724743 . 27934. 1998PNAS...9510564W . free .
Bitinaite . J. . Wah, D. A. . Aggarwal, A. K. . Schildkraut, I. . 1998 . Fok1 dimerization is required for DNA cleavage . Proc Natl Acad Sci USA . 95 . 18 . 10570–5 . 10.1073/pnas.95.18.10570 . 9724744 . 27935. 1998PNAS...9510570B . free .
Tsai, S. Q. et al. (2014). Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576
Guilinger, J. P., Thompson, D. B. & Liu, D. R. (2014). Fusion of catalytically inactive Cas9 to Fok1 nuclease improves the specificity of genome modification. Nature Biotechnol. 32, 577–582
Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K. & Tsai, S. Q. (2015). Dimeric CRISPR RNA-guided Fok1-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431
Strandberg. S.. 2003. Vitamin D receptor start codon polymorphism (Fok1) is related to bone mineral density in healthy adolescent boys. J Bone Miner Metab. 21. 2. 109–13. 12601576. 10.1007/s007740300018. 22436824. etal.