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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.[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, 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.[2]
Its molecular mass is 65.4 kDa, being composed of 587 amino acids.
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 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]
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]
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]