SELECTION OF MUTATIONS ALTERING SPECIFICITY IN RESTRICTION-MODIFICATION ENZYMES USING THE BACTERIOPHAGE-P22 CHALLENGE-PHAGE SYSTEMS

A method for selecting mutants of site-specific DNA-binding proteins h as been applied to the study of the EcoRI and RsrI restriction-modific ation enzymes. Catalytically inactive variants of both endonucleases a re shown to function as pseudo-repressors in the bacteriophage P22 cha llenge-phage assay, and, upon further mutagenesis of the gene encoding R . EcoRI, a variant of that enzyme has been selected which appears t o bind EcoRI-methylated GAATTC sequences to the exclusion of unmethyla ted sites: this specificity is the opposite of that belonging to the n ative enzyme. Variants of the EcoRI methylase have also been found tha t lack either catalytic activity or both binding and catalytic activit ies.