論文

基本情報

氏名 池田 正五
氏名(カナ) イケダ シヨウゴ
氏名(英語) Ikeda Shiyogo
所属 生命科学部 生物科学科
職名 教授
researchmap研究者コード 1000113908
researchmap機関 岡山理科大学

題名

Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes.

単著・共著の別

著者

Ikeda S, Seki S, Watanabe S, Hatsushika M, Tsutsui K.

概要

A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.


発表雑誌等の名称

Analytical Biochemistry

出版者

192

開始ページ

96

終了ページ

103

発行又は発表の年月

1991/01

査読の有無

有り

招待の有無

記述言語

英語

掲載種別

研究論文(学術雑誌)

ISSN

ID:DOI

ID:NAID(CiNiiのID)

ID:PMID

URL

JGlobalID

arXiv ID

ORCIDのPut Code

DBLP ID