Doctor of Philosophy (PhD)
The global conformations of DNA polymerase I from Thermus aquaticus (Taq/Klentaq) and E. coli (Pol I/Klenow) both in isolation and in complex with DNA have been examined in solution using hydrodynamic (analytical ultracentrifugation) and small/wide angle X-ray scattering techniques and then compared to their known crystal structures to assess the similarities and differences in the overall structure of these enzymes and enzyme complexes within a solution environment. These studies address the orientation of the 5’ nuclease domain with respect the polymerase domain (elongated or compact) for the full-length polymerases, and the binding mode (“polymerization” versus “editing”) of the DNA substrate when bound to Klentaq and Klenow. Comparisons of experimental and structure-based data indicate that full-length Pol I and Taq in solution adopt a conformation where the 5’ nuclease domain is extended away from the polymerase domain, similar to the elongated crystal structure. Ab initio shape models of the apo polymerases generated from the scattering data demonstrate remarkable likeness to their corresponding crystal structures and also reveal regions of flexibility. For DNA bound Klenow and Klentaq, comparative analyses indicate that 1) the global conformations of the complexes are not dependent upon the structure of the DNA substrate (primed-template versus blunt-ended) but are polymerase specific, 2) DNA binds to Klenow in the editing mode and to Klentaq in the polymerization mode, and 3) the solution structures deviate somewhat from the crystal structure-based models. Additionally, the stability landscape of Klenow, as monitored by high-throughput thermal denaturation in a variety of solution conditions, demonstrates that Klenow’s melting temperature (Tm) increases with increasing salt concentration and decreasing pH; Klenow’s Tm spans from 40 to 62 degrees C. Both cation and anion specific stabilization is observed. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein. Both the global conformation and the stability studies demonstrate the importance of the solution environment in the comprehensive characterization of an enzyme’s structure and function. The ability to visualize these polymerases and polymerase complexes in solution promises to open new avenues of understanding of these important enzymes.
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Richard, Allison Joubert, "Solution Conformations and Structural Thermodynamics of Type I DNA Polymerases from Escherichia coli and Thermus aquaticus" (2008). LSU Doctoral Dissertations. 65.
Vincent J. LiCata