Pre-Steady State Kinetic Studies of the Fidelity of Nucleotide Incorporation by Yeast DNA Polymerase δ

Abstract

Eukaryotic DNA polymerase delta (pol delta) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol delta is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol delta is one of the major eukaryotic replicative polymerase, the mechanism by which it incorporates nucleotides is unknown. Here we report both steady state and pre-steady state kinetic studies of the fidelity of pol delta. We found that pol delta incorporates nucleotides with an error frequency of 10(-4) to 10(-5). Furthermore, we showed that for correct versus incorrect nucleotide incorporation, there are significant differences between both pre-steady state kinetic parameters (apparent K(d)(dNTP) and k(pol)). Somewhat surprisingly, we found that pol delta synthesizes DNA at a slow rate with a k(pol) of approximately 1 s(-1). We suggest that, unlike its prokaryotic counterparts, pol delta requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.