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Restriction endonuclease Fok1, C terminal
Restriction endonuclease Fok1 bound to DNA PDB 1fok
Identifiers
Symbol	Endonuc-Fok1_C
Pfam	PF09254
Pfam clan	CL0415
InterPro	IPR015334
SCOP2	2fok / SCOPe / SUPFAM
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

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. 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, 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.

DNA-cleavage domain

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

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.

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), or inactive Cas9

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.

References

1. Aggarwal, A. K.; Wah, D. A.; Hirsch, J. A.; Dorner, L. F.; Schildkraut, I. (1997). "Structure of the multimodular endonuclease Fok1 bound to DNA". Nature. 388 (6637): 97–100. doi:10.1038/40446. PMID 9214510. S2CID 205027830.
2. ^ Durai S, Mani M, Kandavelou K, Wu J, Porteus M, Chandrasegaran S (2005). "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Res. 33 (18): 5978–90. doi:10.1093/nar/gki912. PMC 1270952. PMID 16251401.
3. ^ 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. Bibcode:1998PNAS...9510564W. doi:10.1073/pnas.95.18.10564. PMC 27934. PMID 9724743.
4. ^ 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. Bibcode:1998PNAS...9510570B. doi:10.1073/pnas.95.18.10570. PMC 27935. PMID 9724744.
5. Tsai, S. Q. et al. (2014). Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 doi:10.1038/nbt.2908
6. 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 doi:10.1038/nbt.2909
7. 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 doi:10.1089/hum.2015.084
8. Strandberg, S.; et al. (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. doi:10.1007/s007740300018. PMID 12601576. S2CID 22436824.

See also

• Protein engineering
• Genetic engineering
• Zinc finger nuclease

• Bacterial enzymes
• Restriction enzymes