The quaternary structure of DNA polymerase ε from Saccharomyces cerevisiae

Abstract

DNA polymerase ε (Pol ε) trom Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol ε have been cumbersome due to protease sensitivity and the limited amounts of Pol ε in cells. We have developed a protocol for overexpression and purification of Pol ε from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol ε was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol ε of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol ε. Thus, both DNA polymerase δ and Pol ε are purified as monomeric complexes, in agreement with accumulating evidence that Pol δ and Pol ε are located on opposite strands of the eukaryotic replication fork.