Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase δ

Eukaryotic DNA polymerase δ (pol δ) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol δ is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol δ 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 δ. We found that pol δ 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 δ synthesizes DNA at a slow rate with a k_pol of ∼1 s^-1. We suggest that, unlike its prokaryotic counterparts, pol δ requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.