HYBRID RESTRICTION ENZYMES - ZINC-FINGER FUSIONS TO FOK-I CLEAVAGE DOMAIN

A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specifi cities by mutating or engineering existing enzymes. This will avoid th e increasingly arduous task of extensive screening of bacteria and oth er microorganisms for new enzymes. Here, we report the deliberate crea tion of novel site-specific endonucleases by linking two different zin c finger proteins to the cleavage domain of Fok I endonuclease. Both f usion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonu clease and the zinc finger motifs makes it possible to create ''artifi cial'' nucleases that will cut DNA near a predetermined site. This ope ns the way to generate many new enzymes with tailor-made sequence spec ificities desirable for various applications.