FokI nuclease requires __________ for DNA cleavage.
FokI restriction enzyme from Flavobacterium okeanokoites is Type IIS enzyme recognizing GGATG and cutting 9/13 downstream, separated into N-terminal DNA recognition domain and C-terminal cleavage domain. Nuclease domain isolated for genome editing displays catalytic motif PD...D/EXK coordinating Mg2+ for phosphodiester hydrolysis. Critical feature discovered by crystallography is cleavage domain inactive as monomer because catalytic center requires dimerization to align two active sites across DNA duplex forming DSB. Biochemical assays show monomeric FokI binds but cannot incise even with Mg2+. In ZFN context, two monomers must bind in close proximity with spacer length 5-7 bp optimal allowing C-terminal domains to interact via hydrophobic interface. Dimerization requirement provides double checkpoint enhancing specificity, since single ZFN binding does not lead to breakage unlike monomeric nucleases. Engineering obligate heterodimer FokI variants DD: ELAN and RR:DRSH introducing electrostatic repulsion for homodimers further reduces off-target activity, improving therapeutic window. Same principle exploited in TALEN and dimeric Cas9-FokI systems where spatial proximity essential for catalysis ensuring genome stability.
Ref: Bitinaite et al. PNAS 1998 95:10570 FokI dimer; Miller Nat Biotech 2007 Heterodimer; Waugh 1993.