Localization of a protein-DNA interface by random mutagenesis

The type I restriction and modification enzymes do not possess obvious DNA- binding motifs within their target recognition domains (TRDs) of 150-180 am ino acids. To identify residues involved in DNA recognition, changes were m ade in the amino-TRD of EcoKI by random mutagenesis, Most of the 101 substi tutions affecting 79 residues had no effect on the phenotype, Changes at on ly seven positions caused the loss of restriction and modification activiti es. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval be tween residues 80 and 110, Sequence analyses have led to the suggestion tha t the TRDs of type I restriction enzymes include a tertiary structure simil ar to the TRD of the HhaI methyltransferase, and to a model for a DNA-prote in interface in EcoKI, In this model, the residues within the interval iden tified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substi tution impaired both activities. Proteins from twelve mutants were purified : six from mutants with partial or wildtype activity and six from mutants l acking activity. There is a strong correlation between phenotype and DNA-bi nding affinity, as determined by fluorescence anisotropy.