Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit

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 chara...

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Bibliographic Details
Published in:Protein expression and purification 2004-06, Vol.35 (2), p.248-256
Main Authors: Oshige, Masahiko, Takeuchi, Ryo, Ruike, Tatsuji, Ruike, Ryuji, Kuroda, Kazufumi, Sakaguchi, Kengo
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Catalytic Domain
Chromatography, Affinity - methods
DNA Polymerase II - chemistry
DNA Polymerase II - isolation & purification
DNA Polymerase II - metabolism
DNA Primers
Drosophila melanogaster - embryology
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Summary: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%.
ISSN:1046-5928