Using Saccharomyces cerevisiae as a Model to Investigate the Role of Human NAD Kinase

Crystal Ann L. dela Torre
University of Pittsburgh

The interactions of proteins with specific DNA base sequences underlie many important biological processes (e.g. regulation of gene expression). We use the restriction endonuclease EcoRI as a model to understand the structural and energetic factors that determine specificity in protein-DNA interactions. EcoRI exhibits stringent discrimination; it binds and cleaves its DNA recognition site (GAATTC) at least 10,000,000-fold faster than incorrect (miscognate) sites that differ by only one base-pair. This work focuses on thermodynamic and kinetic characterization of a promiscuous mutant EcoRI endonuclease in which a histidine has been substituted for a tyrosine at the protein-DNA interface. The H114Y mutant exhibits relaxed specificity; it cleaves miscognate sites much more efficiently than wild type enzyme, producing lethal effects in vivo. Like the A138T and E192K promiscuous mutants (also being characterized in our laboratory), the H114Y protein binds the cognate recognition site better than the wild type enzyme. Another striking observation is that wild type enzyme achieves binding equilibrium in 20 minutes, whereas the H114Y mutant requires 4.5 hours. This presumably reflects differences between the two enzymes in the intrinsic kinetic constants for wild type and mutant complex formation and suggests a rate-limiting conformational transition for the region in which the histidine resides. Upon complex formation, the proper folding of this region (disordered in the unbound enzyme) must play a crucial role in specificity. Finally, a comparison of the binding and cleavage parameters of wild type and H114Y enzymes at miscognate and nonspecific sites illuminates the basis for in vivo promiscuity and consequent toxicity of the mutant enzyme to host bacterial cells.