Engineering Useful Type I restriction enzymes

DESCRIPTION (provided by applicant): Restriction enzymes are essential laboratory tools for analyzing and cloning DNA. Two principal kinds exist in nature that differ in the way they cleave the substrate. Type II restriction enzymes cleave DNA at fixed positions with respect to their recognition sequences, producing fragments of defined length with predictable termini. These are the useful enzymes, familiar to molecular biologists everywhere. Type I enzymes cleave DNA at random with respect to their recognition sequences, producing fragments of variable length with unpredictable termini. These enzymes are not useful. We propose altering type I enzymes to make them cleave at fixed positions. This will be achieved by replacing the type I subunit that catalyzes random cleavage with a type IIG domain that catalyzes fixed cleavage. The altered enzymes should now cleave close to their recognition sequences, and as a result they should be useful. Since the sequences recognized by type I enzymes are determined by subunits that are modular and appear to be interchangeable, altering just a few enzymes could open the door to a very large number of new specificities, and lead to the fabrication of 'designer' enzymes whose specificities could be custom-tailored, ultimately, to a user's needs. This project will provide molecular biologists with an array of new enzymes for DNA analysis and dissection. The enzymes will be modular, and the DNA sequences to which they attach will change according to which modules are provided. By combining the right modules, sequences of choice might be targeted in living cells with sufficient accuracy to pave the way for gene surgery.