Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase e holoenzyme

Abstract

In eukaryotic DNA replication, DNA polymerase ϵ (Polϵ) is responsible for leading strand synthesis, whereasDNApolymerases α and δ synthesize the lagging strand. The human Pole (hPole) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolϵ function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPole holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPole holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolϵ holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPole holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPole holoenzyme an efficient and faithful replicativeDNApolymerase.