Date of Award




Document Type


Degree Name

Doctor of Philosophy (PhD)


Department of Biomedical Sciences

Content Description

1 online resource (xiii, 195 pages) : color illustrations.

Dissertation/Thesis Chair

Janice D Pata

Committee Members

Paul S Masters, Randall H Morse, Richard P Cunningham, Hongmin Li


DnaE, Kinetics, PolC, Polymerases, Pre-Steady State, Staph, Staphylococcus aureus, DNA polymerases, Chemical kinetics

Subject Categories



In this dissertation, I review the fundamental processes and mechanisms for bacterial DNA replication, especially the mechanisms employed by high-fidelity DNA polymerases to replicate the genome. Unlike the prototypical bacterial system from Escherichia coli which uses a single C-family polymerase, DNA polymerase IIIα (Pol IIIα), to replicate the genome, low-GC content Gram-positive bacteria utilize two essential C-family polymerases, PolC and DnaE. PolC and DnaE work cooperatively to replicate the genome, with DnaE initiating synthesis from RNA-primers and PolC performing the bulk synthesis. Although atomic structures of both PolC and Pol IIIα are available, detailed pre-steady state kinetic analysis of the C-family of polymerases has been sparse. Currently only a single study exists on a truncated version of PolC where two domains have been deleted. Here I present detailed pre-steady state kinetic characterization of the full-length proteins, PolC and DnaE, from the low-GC Gram-positive bacterial pathogen Staphylococcus aureus. Previous characterization of the truncated PolC revealed that it followed a similar kinetic cycle as other polymerases, however it displayed unique regulation of its kinetic cycle through a potential rate-limiting step immediately after chemistry. I confirm this finding in chapter 2 and demonstrate that this rate-limiting step is the release of PPi byproduct from the nucleotide incorporation reaction, which is very slow relative to the rate of chemistry of nucleotide addition. This establishes an equilibrium between binding of the nucleotide and chemistry of phosphodiester bond formation, causing chemistry to be reversible. This is independent of the presence or absence of the β-clamp, or the N-terminal and exonuclease domains. Additionally, I demonstrate that only the next correct nucleotide speeds up the release of PPi of the current nucleotide addition cycle, allowing for fast and processive synthesis. This has never been described before for a DNA-dependent DNA polymerase. Although DnaE plays a limited role in replication, it has been speculated about how the switch from DnaE to PolC occurs. Pre-steady state kinetic characterization of DnaE in chapter 3 shows that it binds a DNA-primed substrate (DNA) and RNA-primed substrate (hybrid) with similar affinity. However, DnaE associates with a hybrid substrate 25-fold faster and dissociates 50-fold faster than on a DNA substrate. Additionally, on a hybrid substrate DnaE displays reversible chemistry and rate-limiting PPi release. Taken together, these results show that DnaE associates with a hybrid substrate faster and adds a few nucleotides before rapidly dissociating. This provides a critical juncture for PolC to take over replication. These findings will be discussed in the following chapters as well as future studies that should be performed.

Included in

Biochemistry Commons