As an undergraduate, I attended the Ohio State University where I worked in the lab of Dr. Zucai Suo studying the kinetic mechanisms of replicative and translesion synthesis DNA polymerases. After receiving my BS in Biochemistry in 2012, I continued research in Dr. Zucai Suo’s lab as a graduate student in the Ohio State Biochemistry Program. I am entering my fifth year of graduate school and my research is now focused on the biochemical and structural characterization of human DNA polymerase epsilon, a major replicative DNA polymerase of unusual size and complexity.
Numerous genetic studies have provided compelling evidence to establish DNA polymerase (Pol) epsilon as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pol epsilon is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′→5′ exonuclease domain common to many replicative polymerases. In addition, Pol epsilon possesses three small subunits with no known catalytic activity that associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of Pol epsilon from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pol epsilon in vitro. However, similar studies of human Pol (hPol) epsilon have been limited by the difficulty of obtaining hPol epsilon in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPol epsilon from insect host cells has allowed for isolation of greater amounts of active hPol epsilon, thus enabling a more detailed kinetic comparison between hPol epsilon and an active N-terminal fragment of the hPol epsilon catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPol epsilon. We observe that the small subunits increase DNA binding by hPol epsilon relative to p261N, but do not significantly affect the nucleotide incorporation rate constant or nucleotide binding affinity. Furthermore, the small subunits do not appear to affect the rate-limiting step of correct nucleotide incorporation. Together, these data suggest that the role of the small subunits in vivo is primarily limited to mediating protein-DNA and protein-protein interactions. Importantly, this study provides the framework for future kinetic investigation of the impact of the other proteins known to interact with Polε at the replication fork.