Genetic Engineering of Taqi Restriction Endonuclease

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This project will employ a new approach to understanding protein function and design. Protein secondary structure elements fold into precise functional regions (domains), which interact in a three dimensional array to provide biological activity. Insertion of two amino acids into a protein causes a small perturbation which has a certain radius of influence within the three dimensional structure. By introducing two-amino acid insertions into the linear sequence, one can scan the length of a protein and determine which regions might comprise functional domains and which regions act as the hinges that connect the domains. This proposal presents a general method for constructing such insertions in proteins by operating at the level of the DNA sequence that codes for the TagI restriction endonuclease. The aim is to understand the specificity of DNA sequence recognition by TagI endonuclease, and to engineer mutants with altered sequence recognition. Wild type endonuclease, a so called "allosteric activation mutant", and a "canonical site nicking mutant" will be overproduced, purified, and characterized in vitro with respect to sequence specific binding, dissociation, facilitated diffusion, and cleavage, using a variety of DNA substrates. Using a unique TagI endonuclease clone, additional two-codon insertion mutations will be introduced into the enzyme, and characterized by both in vitro and in vitro assays. These studies will help to define the sequence specificity domains, and allow for further targeted saturation mutagenesis of those domains. The new recognition sequence of these mutant proteins will be determined using a uniquely labeled DNA substrate. In collaboration with Dr. John Anderson, purified TagI will be co-crystalized with its cognate oligonucleotide for determination of its three dimensional structure.