title: Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit.
abstract: gfLittle 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. 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 second subunit (Drosophila pol. 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. catalytic subunit as follows: Drosophila pol. 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%.
authors.affiliation: Frontier Research Center for Genome and Drug Research, Tokyo University of Science, Ymazaki 2641, Noda-shi, Chiba-ken 278-8510, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.forename: Ryo
authors.initials: R
authors.lastname: Takeuchi
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Ruike
authors.forename: Ryuji
authors.initials: R
authors.lastname: Ruike
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Kuroda
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002846:Chromatography, Affinity / Q000379:methods*; D004257:DNA Polymerase II / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000196:embryology
chemical_list: D017931:DNA Primers; D004257:DNA Polymerase II