title: The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites.
abstract: The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.
authors.affiliation: The DNA-Proteins Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 4DL, UK. s.halford@bristol.ac.uk
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Lucy E
authors.initials: LE
authors.lastname: Catto
authors.forename: Christian
authors.initials: C
authors.lastname: Pernstich
authors.forename: David A
authors.initials: DA
authors.lastname: Rusling
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Sanders
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D016335:Zinc Fingers / Q000502:physiology
chemical_list: D004247:DNA; D003851:Deoxyribonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific