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

Journal Article · Tue Jan 01 04:00:00 EST 1991 · Analytical Biochemistry; (United States)

DOI:https://doi.org/10.1016/0003-2697(91)90191-U· OSTI ID:5557070

Ikeda, S; Seki, S; Watanabe, S; Hatsushika, M; Tsutsui, K ^[1]

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1. Department of Biochemistry, Okayama University Medical School (Japan)

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-{sup 32}P-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.

• OSTI ID: 5557070
• Journal Information: Analytical Biochemistry; (United States), Journal Name: Analytical Biochemistry; (United States) Vol. 192:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
ANTI-INFECTIVE AGENTS
ANTIBIOTICS
ANTIMITOTIC DRUGS
ANTINEOPLASTIC DRUGS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
BLEOMYCIN
DAYS LIVING RADIOISOTOPES
DNA
DNA POLYMERASES
DNA REPAIR
DRUGS
ELECTROPHORESIS
ENZYMES
FRACTIONATION
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANES
MICE
MOLECULAR WEIGHT
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
RECOVERY
REPAIR
RODENTS
SEPARATION PROCESSES
TRANSFERASES
TUMOR CELLS
VERTEBRATES