STRUCTURE OF THE MULTIMODULAR ENDONUCLEASE FOKI BOUND TO DNA

FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence(1-3) Because of its unusual b ipartite nature, FokI has been used to create artificial enzymes with new specificities(4-7). We have determined the crystal structure at 2. 8 Angstrom resolution of the complete FokI enzyme bound to DNA. As ant icipated, the enzyme contains amino- and carboxy-terminal domains corr esponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 an d D3) which are evolutionarily related to the helix-turn-helix-contain ing DNA-binding domain of the catabolite gene activator protein CAP(8) . The CAP core has been extensively embellished in the first two subdo mains, whereas in the third subdomain it has been co-opted for protein -protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. Th e structure suggests a new mechanism for nuclease activation and provi des a framework for the design of chimaeric enzymes with altered speci ficities.