Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.
A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.
Fisher
E W
EW
Department of Biochemistry, College of Medicine, School of Chemical Sciences, University of Illinois at Urbana-Champaign 61801-3602, USA.
Yang
M T
MT
Jeng
S T
ST
Gardner
J F
JF
Gumport
R I
RI
D017100:Bacteriophage P22 / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D014644:Genetic Variation; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)