A site-specific endonuclease derived from a mutant Trp repressor with altered 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" alpha-helix of the helix-turn-helix DNA-binding motif. Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors. Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot. One mutant 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 repressor. A site-specific nuclease was derived from Lys79 repressor by constructing 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 vitro. When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency.
Pfau
J
J
California Institute of Biological Research, La Jolla 92037, USA.
Arvidson
D N
DN
Youderian
P
P
Pearson
L L
LL
Sigman
D S
DS
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D003851:Deoxyribonucleases; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins