A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The Trp repressor controls gene expression in response to tryptophan levels.
• The helix-turn-helix motif is crucial for DNA binding in many regulatory proteins.
• Understanding repressor-operator interactions is key to gene regulation.

Purpose of the Study:

• To investigate the role of amino acid residues Ile79 and Ala80 in the Trp repressor's DNA-binding helix.
• To generate mutant Trp repressors with altered DNA-binding properties and specificities.
• To engineer a site-specific nuclease based on a modified Trp repressor.

Main Methods:

• Site-directed mutagenesis was employed to create single amino acid substitutions at positions Ile79 and Ala80.
• Mutant repressors were analyzed for altered activity, DNA-binding specificity, and operator interaction in vivo.
• A double-mutant protein (Lys79/Cys49) was generated and modified with a phenanthroline-copper adduct to create a nuclease.

Main Results:

• Eight mutant repressors exhibited super-aporepressor activity under tryptophan-limiting conditions.
• Eleven mutants displayed extended DNA-binding specificities, recognizing operators not bound by wild-type repressor.
• The Lys79 mutant showed altered specificity, binding a mutant operator more effectively than the wild-type operator.
• The derived Lys79 nuclease demonstrated altered DNA binding in vitro and efficient, specific cleavage of operator DNA upon activation.

Conclusions:

• Amino acid residues Ile79 and Ala80 significantly influence Trp repressor DNA-binding specificity and activity.
• Targeted mutagenesis can generate repressors with novel DNA-binding properties and create engineered nucleases.
• The Lys79-derived nuclease represents a tool for site-specific DNA cleavage with altered recognition properties.