Poster abstracts
Poster number 68 submitted by Walter Zahurancik
Kinetic characterization of the human DNA polymerase ε holoenzyme
Walter Zahurancik (Ohio State Biochemistry Program, Department of Chemistry and Biochemistry)
Abstract:
Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ 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, Polɛ 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 Polɛ from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of human Polɛ (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ 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.
Keywords: human DNA polymerase epsilon, DNA replication, pre-steady-state kinetics