The quaternary structure of DNA polymerase e from Saccharomyces cerevisiae
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 16, 14082-14086 p.Article in journal (Refereed) Published
DNA polymerase e (Pol e) trom Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol e have been cumbersome due to protease sensitivity and the limited amounts of Pol e in cells. We have developed a protocol for overexpression and purification of Pol e from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol e 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 e 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 e. Thus, both DNA polymerase d and Pol e are purified as monomeric complexes, in agreement with accumulating evidence that Pol d and Pol e are located on opposite strands of the eukaryotic replication fork.
Place, publisher, year, edition, pages
2003. Vol. 278, no 16, 14082-14086 p.
Engineering and Technology
IdentifiersURN: urn:nbn:se:liu:diva-46660DOI: 10.1074/jbc.M211818200OAI: oai:DiVA.org:liu-46660DiVA: diva2:267556