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Restriction enzyme From Wikipedia, the free encyclopedia
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.[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 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,[3] producing two sticky ends with 4-bp overhangs.
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.[3]
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]
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.[4]
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),[2] or inactive Cas9[5][6][7]
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.[8]
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