The restriction endonuclease Fok1, naturally found in ''

Flavobacterium ''Flavobacterium'' is a genus of Gram-negative, nonmotile and motile, rod-shaped bacteria that consists of 130 recognized species. Flavobacteria are found in soil and fresh water in a variety of environments. Several species are known to cause ...

okeanokoites'', is a bacterial type IIS

restriction endonuclease A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

consisting of an N-terminal

DNA-binding domain A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a gener ...

and a non sequence-specific DNA cleavage domain at the

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

. 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

nucleotide Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...

s 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 The molecular mass () is the mass of a given molecule, often expressed in units of daltons (Da). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The derived quan ...

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 A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) which stabilizes the fold. The term ''zinc finger'' was originally coined to describe the finger-like appearance of a ...

(see zinc finger nuclease), or inactive

Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 dalton (unit), kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utili ...

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 One of several human vitamin D receptor gene variants is caused by a

single nucleotide polymorphism In genetics and bioinformatics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in ...

in the start codon of the gene which can be distinguished through the use of the Fok1 enzyme.

DNA-binding domain

DNA-cleavage domain

Activity

Exploitation

References

See also