Ew. Fisher et al., SELECTION OF MUTATIONS ALTERING SPECIFICITY IN RESTRICTION-MODIFICATION ENZYMES USING THE BACTERIOPHAGE-P22 CHALLENGE-PHAGE SYSTEMS, Gene, 157(1-2), 1995, pp. 119-121
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.