Title page for ETD etd-11112009-121622


Type of Document Dissertation
Author Wowor, Andy James Budiman
Author's Email Address awowor1@lsu.edu
URN etd-11112009-121622
Title DNA Structural Selectivity of Binding by the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus
Degree Doctor of Philosophy (Ph.D.)
Department Biochemistry (Biological Sciences)
Advisory Committee
Advisor Name Title
LiCata, Vince J. Committee Chair
Aboul-ela, Fareed M. Committee Member
Grove, Anne Committee Member
Siebenaller, Joseph F. Committee Member
Constant, W. David Dean's Representative
Keywords
  • Analytical Ultracentrifugation
  • Circular Dichroism
  • Thermodynamics
  • Ion Linkage
  • Binding Constants
  • Heat Capacity Change
  • Protein-DNA Interaction
  • Escherichia coli
  • DNA Polymerase
  • Thermus aquaticus
  • Pol I
  • Taq Polymerase
  • Klenow
  • Klentaq
  • DNA
  • DNA Structure
  • Single Stranded
  • Primer Template
  • Blunt End
  • Electrophoretic Mobility Shift Assay
  • Isothermal Titration Calorimetry
  • Structural Selectivity
  • Fluorescence Anisotropy
Date of Defense 2009-07-06
Availability unrestricted
Abstract
Understanding the thermodynamics of substrate selection by DNA Polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the “large fragments” of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologues. Klentaq, however, does not have a functional proofreading site.

Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can differ significantly at any individual salt concentration. For both proteins, binding of ss-DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release 2-3 more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Both of these non-sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding, however, Klenow exhibits significant differences in the ΔCp of binding to pt-DNA versus ds-DNA, while Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the ΔG, ΔCp, and salt dependence patterns suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.

Structural data from the electrophoretic mobility shift assay (EMSA) also support a striking difference between ds-DNA binding for Klenow and Klentaq. In EMSA, all ds-DNA/Klenow complexes show a time dependent shift from a slower to a faster moving complex while pt-DNA/Klenow complexes (both matched and mismatched) are found only in the fast moving complex. In contrast, all DNA/Klentaq complexes are observed in a slower moving complex only. Several potential molecular models for correlating the thermodynamics and the structural data are discussed. The thermodynamic differences among the different DNA structural preferences for the two polymerases suggest that the in vivo functions of these two largely homologous polymerases are somewhat different and respond differently to environmental conditions.

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