The long term objective is to understand sequence-specific recognition of DNA by proteins using restriction-modification (RM) enzymes as simple model systems. The plan here is to study the FokI RM system. FokI is a Type IIS restriction endonuclease which recognizes the pentanucleotide duplex, GGATG CATCC and cleaves about 9/13 bp away from the recognition site. This implies that presence of two separate protein domains in this enzyme: one for sequence-specific recognition and the other for the endonuclease activity. Once the DNA binding domain anchors at the appropriate site, a signal is transmitted to the endonuclease probably through allosteric interactions and the cleavage occurs. Studies on proteolytic fragments of FokI endonuclease have defined an N-terminal DNA-binding domain and C-terminal domain with non-specific cleavage activity PNAS 89:4275-4279 (1991) . These results have been confirmed by the study of the C-terminal deletion mutants of FokI endonuclease Gene 133:79-84 (1993) . Furthermore, introduction of additional amino acid residues between the recognition and cleavage domains of FokI can alter the cleavage distance from the recognition site within its DNA substrate PNAS 90:2764-2768 (1993) . These studies suggest that the two domains of FokI are connected by a linker region which appears to be amenable for repositioning of the DNA-binding domain with respect to the catalytic domain. More recently, successful engineering of the first chimeric restriction endonuclease by linking the Ubx homeodomain to the catalytic domain of (FN) of FokI was reported PNAS 91:883-887 (1994) . The objective here is to continue the study on the FokI RM system. The goal here is to construct several additional C-terminal deletion variants of FokI to precisely define the recognition domain and linker/catalytic domain and to engineer chimeric restriction enzymes with novel sequence-specificities by linking other DNA binding proteins (e.g. zinc finger motifs) to the cleavage domain of FokI endonuclease. The aim is also to alter the sequence-specificity of FokI recognition domain by random mutagenesis and affinity selection of mutants with altered sequence-specificities using the phage display system; and hence alter the sequence specificity of FokI endonuclease. This work could lead to the development of an array of artificial nucleases with tailor-made sequence-specificities desirable for various applications. %%% Since their discovery nearly 25 years ago, Type II restriction enzymes have played a crucial role in the development of the recombinant DNA technology and the field of molecular biology. A long term goal in the field of RM enzymes has been to generate restriction endonucleases wi th novel sequence- specificities by mutating or engineering existing enzymes. Our studies on proteolytic fragments of FokI endonuclease (belonging to the Type IIS class) have revealed an N-terminal DNA-binding domain and a C-terminal domain with nonspecific DNA-cleavage activity. The modular structure of FokI endonuclease suggested that it might be feasible to construct hybrid endonucleases with novel sequence- specificities by linking other DNA-binding proteins to the cleavage domain. Recently, we reported the construction of the first `chimeric` restriction endonuclease by linking Ubx homeodomain to the cleavage domain of FokI. Our goal here is continue our work on the hybrid endonucleases. This work could lead to the development of an array of artificial nucleases with tailor-made sequence- specificities desirable for various applications.
