FokI (biology)

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.^[1] Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3': 5'-CATCC-3' recognition site, the DNA cleavage domain is activated and cleaves non-specifically between nine and 13 nucleotides downstream of the recognition site.^[2]

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

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

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).^[1]

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.^[4]

References

1. ^ ^{a b} 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. PMID 16251401.
2. ^ ^{a b c} Wah, D. A.; J. Bitinaite, Schildkraut, I., Aggarwal, A. K. (1998). "Structure of FokI has implications for DNA cleavage". Proc Natl Acad Sci U S A 95 (18): 10564-9.
3. ^ ^{a b} Bitinaite, J.; D. A. Wah, Aggarwal, A. K., Schildkraut, I. (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci U S A 95 (18): 10570-5.
4. ^ Strandberg, S.; et al. (2003). "Vitamin D receptor start codon polymorphism (FokI) is related to bone mineral density in healthy adolescent boys". J Bone Miner Metab. 21 (2): 109-13. PMID 12601576.

See also

• Protein engineering
• Genetic engineering
• Zinc finger nuclease