In VivoReconstitution ofSaccharomyces cerevisiaeDNA Polymerase ε in Insect Cells
Journal of Biological Chemistry
DNA polymerase ⑀ (pol ⑀) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol ⑀ has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol ⑀ complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol ⑀ complex and truncated Pol2p (140 kDa), as was observed initially with the pol ⑀
... d from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol ⑀ is functionally identical to native pol ⑀ purified from yeast and is therefore useful for biochemical characterization of the interactions of pol ⑀ with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.