In situ activity gel for DNA repair 3'-phosphodiesterase.
An enzyme that plays an important role in the repair of oxidative DNA damage is the 3'-phosphodiesterase. This activity, which repairs damaged DNA 3'-termini,can be detected using several available biochemical assays. We present a method to detect 3'-phosphodiesterase activity of renatured proteins immobilized in polyacrylamide gels. The model substrate, labeled with [alpha-32P]dCTP, contains 3'-phosphoglycolate termini produced by bleomycin-catalyzed cleavage of the self-complementary alternating copolymer poly(dGdC). The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE. Active 3'-phosphodiesterase enzymes are detected visibly by the loss of radioactivity at a position corresponding to the mobility of the enzyme during SDS-PAGE. Using this procedure, two Escherichia coli 3'-phosphodiesterases, exonuclease III and endonuclease IV, are readily detected in crude cell extracts or as homogeneous purified proteins. Extracts of mutant cells lack activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (Mr approximately 100 kDa). The identification of this novel 100 kDa E.coli 3'-phosphodiesterase demonstrates the potential value of the activity gel method described here.
Sander
M
M
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, POB 12233, Research Triangle Park, NC 27709, USA. sander@niehs.nih.gov
D004249:DNA Damage; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D018384:Oxidative Stress; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012680:Sensitivity and Specificity
D010727:Phosphoric Diester Hydrolases