Poster abstracts
Poster number 56 submitted by Walter Zahurancik
Pre-steady-state kinetic characterization of human DNA polymerase ε
Walter J. Zahurancik (The Ohio State Biochemistry Program), Seth J. Klein (Department of Molecular Genetics, The Ohio State University), Zucai Suo (The Ohio State Biochemistry Program)
Abstract:
Maintenance of eukaryotic genome stability requires the action of DNA polymerases (Pols) that are capable of replicating genomic DNA efficiently and accurately. Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to an overall low polymerization error frequency (one error per 106-108 incorporations) and support faithful eukaryotic genome replication. Three replicative DNA polymerases from the B-family, Polα, Polδ, and Polε, are responsible for the majority of eukaryotic DNA replication. Polε has been shown to catalyze leading-strand DNA synthesis during DNA replication in vivo, but little is known about the kinetic mechanism and overall fidelity of polymerization catalyzed by this replicative enzyme. We have utilized pre-steady-state kinetic methods to elucidate the mechanism of DNA polymerization catalyzed by human Polε (hPolε) and provide a kinetic basis for high fidelity DNA replication. Our results show that hPolε follows an induced-fit mechanism by which it exhibits large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides, resulting in a low frequency of mismatched base pair formation (10-4-10-7 per nucleotide incorporation event). The 3′→5′ exonuclease activity of hPolε further enhances polymerization fidelity by an unprecedented 3.5x102 to 1.2x104-fold. With an overall fidelity of 106-108 (0.1-1.0 error per round), hPolε is conclusively justified in being the primary enzyme to replicate the human nuclear genome. Consistently, somatic mutations in hPolε, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.
References:
Zahurancik, W. J., Klein, S.J., Suo, Z.* (2014) Significant Contribution of the 3’→5′ Exonuclease Activity to the High Fidelity of Nucleotide Incorporation Catalyzed by Human DNA Polymerase ε. Nucleic Acids Research. 42 (22), 13853-60.
Zahurancik, W.J., Klein, S.J., Suo, Z.* (2013) Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε, Biochemistry. 52, 7041−7049.
Keywords: Pre-steady-state kinetics, Human DNA polymerase epsilon, 35 exonuclease activity