Doctor of Philosophy (PhD)
This study examines several different linkages between the nucleic acid binding thermodynamics and the functions of three different DNA polymerases. The focuses are correlation of the DNA binding thermodynamics and the functional behavior of Klenow and Klentaq polymerases (from Escherichia coli and Thermus aquaticus, respectively), identification of factors that influence the proofreading activity of Klenow, and examination of HIV reverse transcriptase (HIV-RT) binding to different primer/template nucleic acid constructs. A comparison of the DNA binding thermodynamics and the incorporation activity of Klenow and Klentaq reveals that the enthalpic versus entropic balance upon binding may function as a modulator of the temperature dependence of the enzymatic activity. Both polymerases bind DNA with nanomolar affinity at significantly low temperatures, but have negligible enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding crosses zero and becomes favorable (negative). Proofreading activity improves the fidelity of DNA synthesis. Proofreading requires unwinding of the primer strand and shuttling of the 3’ terminus of the primer from the polymerization site to the proofreading site. The binding of Klenow to matched and mismatched primed-template DNA was examined by monitoring the steady state fluorescence intensity change of a 2-aminopurine base site-specifically substituted in DNA and reveals that both the equilibrium partitioning and the dynamic partitioning between sites are dependent on the absence, presence, and identity of specific divalent cations, as well as on the presence of mismatched bases at the primer/template junction. HIV-RT performs both DNA and RNA template directed DNA synthesis. Direct binding equilibria have been characterized for the interaction of HIV-RT with several different primer/template nucleic acid constructs across a range of KCl concentrations. The thermodynamic affinities of the two homoduplexes (DNA/DNA and RNA/RNA) are shown to be nearly identical, while binding of the heteroduplexes is significantly tighter. At least two different modes of nucleic acid binding are revealed by the thermodynamic salt linkages of binding, and these different thermodynamic binding modes correlate with different recently structurally elucidated binding modes.
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Brown, Hiromi Shimasaki, "Nucleic Acid Binding Thermodynamics and Functions of DNA Polymerases" (2013). LSU Doctoral Dissertations. 2650.