The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal alpha helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.

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. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, 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. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.

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Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.