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Title page for ETD etd-07222008-151325


Type of Document Dissertation
Author Weiner, Brian Edward
Author's Email Address brian.weiner@vanderbilt.edu
URN etd-07222008-151325
Title Biochemical and structural analysis of the p58C and p68N domains of DNA polymerase alpha/primase
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Walter J. Chazin Committee Chair
Brandt F. Eichman Committee Member
David Cortez Committee Member
Ellen Fanning Committee Member
Richard N. Armstrong Committee Member
Keywords
  • DNA polymerases -- Structure
  • T antigen
  • DNA primase
  • DNA polymerase alpha
  • DNA replication
  • SV40
  • NMR solution structure
Date of Defense 2008-06-09
Availability unrestricted
Abstract
The replication of DNA occurs through a complex series of steps involving the coordinated action of many proteins. DNA polymerase alpha/primase (pol-prim) is a critical DNA replication factor that synthesizes short RNA-DNA primers on the leading and lagging strands of DNA being actively replicated. Pol-prim contains a DNA polymerase subunit (p180), an accessory subunit (p68), and two DNA primase subunits (p58 and p48). This dissertation describes the biochemical and structural characterization of two folded, globular pol-prim domains: p58C and p68N. Sub-cloning, bacterial expression, purification, and analysis of p58C revealed an essential iron-sulfur cluster, the first such cofactor identified in a DNA replication protein. Sub-cloning, bacterial expression, and NMR analysis enabled the determination of the solution structure of p68N. The binding of p68N to the SV40 large T antigen helicase domain was measured by isothermal titration calorimetry and the structure was used to model and test the molecular basis for this interaction. These results enabled an updated model to be generated for the action of pol-prim in SV40 DNA replication.
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