Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes.
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.
Ikeda
S
S
Department of Biochemistry, Okayama University Medical School, Japan.
Seki
S
S
Watanabe
S
S
Hatsushika
M
M
Tsutsui
K
K
D000818:Animals; D001761:Bleomycin / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases; D008567:Membranes, Artificial; D051379:Mice; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D012967:Sodium Dodecyl Sulfate; D014407:Tumor Cells, Cultured / Q000187:drug effects
D008567:Membranes, Artificial; D010761:Phosphorus Radioisotopes; D001761:Bleomycin; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III