FokI requires two specific DNA sites for cleavage

FokI is a bipartite restriction endonuclease that recognizes a non-palindro mic DNA sequence, and then makes double-stranded cuts outside of that seque nce to leave a 5' overhang. Earlier kinetic and crystallographic studies su ggested that FokI might function as a dimer. Here, we show, using dynamic l ight-scattering, gel-filtration and analytical ultracentrifugation, that Fo kI dimerizes only in the presence of divalent metal ions. Furthermore, anal ysis of the DNA-bound complex reveals that two copies of the recognition se quence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have b road implications for the mechanism by which monomeric type II endonuclease s achieve high fidelity. (C) 2001 Academic Press.