A Non-radioactive, Zymographical Method for Detecting Mammalian DNA Repair Enzymes
A non-radioactive zymographical method for detecting mammalian DNA repair enzymes is presented. The method consists of the following steps: (1) preparations of crude, partially purified enzymes; (2) SDS-(denatured) polyacrylamide gel electrophoresis (PAGE) of the enzyme preparations; (3) renaturation of proteins electrophoresed on SDS-PAGE; (4) preparation of damaged DNA-fixed membranes; (5) protein blotting (activity blotting) onto a damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by a repair enzyme(s); (6) non-radioactive detection of the activity-blotted site(s) to localize the repair enzyme. The present technique was developed using mouse APEX nuclease, a multifunctional DNA repair enzyme, and a DNA-fixed membrane treated with bleomycin or acid-depurinated, and was applied for the detection of human liver APEX nuclease in partially purified preparations. The sites primed on damaged DNA by APEX nuclease, which has priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA produced by generating free 3'-hydroxyl termini, were labeled with digoxygenin-11-dUTP by incubation with a digoxygenin-labeled substrate and Klenow polymerase. The digoxygenin-labeled DNA on the activity-blotted membrane was detected by a sensitive chemiluminescent reaction. Human liver APEX nuclease in a partially purified preparation and an active peptide possibly derived from the enzyme were clearly demonstrated by the present method.