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Multiequilibria of Oligomeric Thermophilic DNA Replication Polymerases

Lin, Hsiang-Kai (2013) Multiequilibria of Oligomeric Thermophilic DNA Replication Polymerases. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. We examined the thermodynamics and enzymatic activity related to the oligomerization of hyperthermophilic archaeal Sulfolobus solfataricus (Sso) primary DNA replication polymerase (Dpo1) and lesion bypass polymerase (Dpo4). Both Dpo1 and Dpo4 bind to DNA with initial high affinity monomeric binding followed by sequential binding of additional molecules at higher concentrations of the enzyme. Gel filtration, chemical crosslinking, isothermal titration calorimetry (ITC) and fluorescence anisotropy experiments all show a stoichiometry of three Dpo1 and two-four Dpo4 molecules bound to a single DNA substrate. In particular, oligomeric Dpo1-DNA complexes significantly increase both the kinetic rate and processivity of DNA synthesis.
Differentiation of binding accurate DNA replication polymerase Dpo1 over error prone DNA lesion bypass polymerase Dpo4 is essential for the proper maintenance of the genome. Binding discrimination between these polymerases on DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Fluorescence anisotropy experiments were used to separate discrete binding events for the formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature-dependent, generally leading to more favorable binding at higher temperatures for both polymerases. At high temperatures, DNA binding of Dpo1 monomer is slightly favored over binding of Dpo4 monomer, but binding of Dpo1 trimer is strongly favored over binding of Dpo4 dimer, thus providing thermodynamic selection.
The results from ITC showed an unusually strong temperature dependence of the change in heat capacity (∆C_p^o), which switches from positive to negative values with increasing temperature. The observed sign change in ∆C_p^o does not derive from temperature-dependent changes in structure, protonation, or electrostatics. Rather, we propose that temperature affects the coupled equilibria between self-associations of free Dpo1 or Dpo4 and their binding to DNA. Taken together, Sso differentiates between Dpo1 and Dpo4 binding to DNA by integrating molecular and cellular principles including concentration, temperature, oligomerization, and coupled equilibria to maintain uninterrupted, rapid, and high fidelity DNA replication.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Lin, Hsiang-Kaihsl15@pitt.eduHSL15
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairTrakselis, Michaelmtraksel@pitt.eduMTRAKSEL
Committee MemberJen-Jacobson, Lindaljen@imap.pitt.eduLJEN
Committee MemberSaxena, Sunilsksaxena@pitt.eduSKSAXENA
Committee MemberHorne, Sethhorne@pitt.eduHORNE
Date: 18 October 2013
Date Type: Publication
Defense Date: 6 August 2013
Approval Date: 18 October 2013
Submission Date: 4 September 2013
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 190
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: thermodynamics, polymerase,oligomerization,coupled equilibrium, Sulfolobus solfataricus, archaeal, heat capacity change, temperature dependent
Date Deposited: 18 Oct 2013 13:58
Last Modified: 18 Oct 2018 05:15


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