Yeast DNA polymerase ε catalytic core and holoenzyme have comparable catalytic rates

Abstract

The holoenzyme of yeast DNA polymerase ε (Pol ε) consists of four subunits: Pol2, Dpb2, Dpb3, and Dpb4. A protease-sensitive site results in an N-terminal proteolytic fragment of Pol2, called Pol2core, that consists of the catalytic core of Pol ε and retains both polymerase and exonuclease activities. Pre-steady-state kinetics showed that the exonuclease rates on single-stranded, double-stranded, and mismatched DNA were comparable between Pol ε and Pol2core. Single-turnover pre-steady-state kinetics also showed that the kpol of Pol ε and Pol2core were comparable when preloading the polymerase onto the primer-template before adding Mg2+ and dTTP. However, a global fit of the data over six sequential nucleotide incorporations revealed that the overall polymerization rate and proces-sivity were higher for Pol ε than for Pol2core. The largest difference between Pol ε and Pol2core was observed when challenged for the formation of a ternary complex and incorporation of the first nucleotide. Pol ε needed less than 1s to incorporate a nucleotide, but several seconds passed before Pol2core incorporated detectable levels of the first nucleotide. We conclude that the accessory subunits and the C terminus of Pol2 do not influence the catalytic rate of Pol ε but facilitate the loading and incorporation of the first nucleotide by Pol ε.