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Title page for ETD etd-02122019-123658

Type of Document Dissertation
Author Holt, Marilyn Elaine
URN etd-02122019-123658
Title Contributions to template priming from the human DNA primase 4Fe-4S cluster domain
Degree PhD
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
Charles R. Sanders Committee Chair
Brandt F. Eichman Committee Member
Jared T. Nordman Committee Member
Katherine L., Friedman Committee Member
  • structural biology
  • molecular biophysics
  • primase
  • DNA replication
  • NMR
  • x-ray crystallography
  • fluorescence spectroscopy
  • SAXS
  • charge transport
  • iron-sulfur cluster
Date of Defense 2018-07-31
Availability restricted
Replication of our genes requires unwinding of DNA and generating the new complementary strands for both leading and lagging parental strands. The DNA polymerases that generate the complementary strands require a short (8-12 nucleotide) “primer” on the template strand, which is synthesized by the DNA-dependent RNA polymerase DNA primase. Human DNA primase is a heterodimer of catalytic (p48) and regulatory (p58) subunits, with a distinct DNA-binding and 4Fe-4S cluster-containing domain in the regulatory subunit (p58C). Both the p58C domain and the 4Fe-4S cluster are critical for proper function of primase, but the specific reasons why are not known. X-ray crystal structures of primase have informed models for the mechanism of catalysis; however, the underlying molecular basis of how primase functions remain unclear.

This dissertation research focused on developing a coherent model for how human DNA primase synthesizes the first dinucleotide, extends the primer, and hands off the substrate. Combining biophysical approaches with collaborative electrochemical studies, we showed that the 4Fe-4S cluster acts as a redox switch to modulate DNA binding affinity and identified a possible charge transfer pathway from the cluster to the DNA binding site. Biochemical and structural characterization of wild-type and charge transfer-deficient mutants revealed very similar structures yet substantial differences in primase activity and product distribution, suggesting a role for this redox switch in primer initiation and counting. Additionally, small-angle X-ray scattering (SAXS) was used to investigate the domain architecture of primase free, when synthesizing the first dinucleotide, and when bound to a full primed substrate. SAXS data acquired under these conditions revealed primase transiently oligomerizes and that it is extended when bound to primed substrate. Our results are consistent with a model whereby p58C travels with the 5’ end of the nascent primer in the context of full-length primase.

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