A long term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence-specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of brute-force screening of bacteria and other microorganisms for new enzymes. Our objective here is to generate rare cutters that would be valuable tools in genomic research and analysis. Over the past six years, we have studied FokI restriction endonuclease from Flavobacterium Okenokoites in great detail. FokI is a Type IIS endonuclease which recognizes the pentanucleotide duplex, 5'-GGATG-3': 5'-CATCC-3' and cleaves about 9/13 bp away from the recognition site. This implies the presence of two separate protein domains in this enzyme: one for sequence-specific recognition and the other for the endonuclease activity. Our studies o 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 athe cleavage distance from the recognition site within its DNA substrate [PNAS 90:2764-2768 (1993); J. Biol Chem.269:31978-31982 (1994)]. These results suggest that the two domains of FokI are connected by a liner region which appears to be amenable for repositioning of the DNA-binding domain with respect to the catalytic domain. Recently, we have successfully engineered th first chimeric restriction endonuclease by linking the Ubx homeodomain to the catalytic domain of (Fn) of FokI [PNAS 91:883-887 (1994)]. More recently, we reported the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of FokI endonuclease. Both fusions are active. This opens the way to generate many new enzymes with tailor-made sequence-specificities [Science, manuscript submitted for publication (1995)]. This work could lead to the development of an array of artificial nucleases with tailor- made sequence-specificities desirable for various applications.
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