Poster abstracts

Poster number 60 submitted by Walter Zahurancik

Kinetic Mechanism of DNA Polymerization Catalyzed by Human DNA Polymerase ε

Walter J. Zahurancik (Chemistry and Biochemistry), Seth J. Klein (Molecular Genetics), Zucai Suo (Chemistry and Biochemistry)

Abstract:
Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a KdDNA of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s−1. Although Polε exo- binds a correct incoming nucleotide weakly with a KddTTP of 31 μM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s−1 at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity.

References:
Zahurancik, W. J., Klein, S. J., and Suo, Z. (2013) “Kinetic Mechanism of DNA Polymerization Catalyzed by Truncated Human DNA Polymerase ε”, Biochemistry. 52, 7041-7049.

Keywords: DNA polymerase , Mechanism, Pre-steady-state