A SITE-SPECIFIC ENDONUCLEASE DERIVED FROM A MUTANT TRP REPRESSOR WITHALTERED DNA-BINDING SPECIFICITY

Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second ''recognition'' al pha-helix of the helix-turn-helix DNA-binding motif. Eight of these mu tant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepres sors. Eleven mutant repressors have extended DNA-binding specificies i n vivo, and bind operators which the wild-type repressor cannot. One m utant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type represso r. A site-specific nuclease was derived from Lys79 repressor by constr ucting a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct. This nuclease has an altered specificity of DNA binding in vi tro. When activated by the addition of thiol and hydrogen peroxide, th e Lys79 nuclease cleaves operator DNA within its new recognition seque nce with high efficiency.