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 damag ed DNA 3'-termini, can be detected using several available biochemical assays, We present a method to detect 3'-phosphodiesterase activity o f renatured proteins immobilized in polyacrylamide gels, The model sub strate, labeled with [alpha-P-32]dCTP, contains 3'-phosphoglycolate te rmini produced by bleomycin-catalyzed cleavage of the self-complementa ry alternating copolymer poly(dGdC). The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE, Active 3'-phosphodieste rase enzymes are detected visibly by the loss of radioactivity at a po sition corresponding to the mobility of the enzyme during SDS-PAGE. Us ing this procedure, two Escherichia coli 3'-phosphodiesterases, exonuc lease III and endonuclease IV, are readily detected in crude cell extr acts or as homogeneous purified proteins, Extracts of mutant cells lac k activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (M-r approxi mate to 100 kDa). The identification of this novel 100 kDa E. coli 3'- phosphodiesterase demonstrates the potential value of the activity gel method described here.