Abstract
Little is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol.epsilon catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase epsilon second subunit (Drosophila pol.epsilon 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol.epsilon catalytic subunit as follows: Drosophila pol.epsilon catalytic subunit synthesized DNA processively in the presence of both Mn[2+] and Mg[2+] ions, but Mn[2+] inhibited the 3'–5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.
