Contributions to template priming from the human DNA primase 4Fe-4S cluster domain

Abstract

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.