Figure 4.

H₂O₂ stimulates TOP2- but not TOP1-mediated DNA cleavage. (A) Stimulation of calf thymus TOP2-mediated DNA cleavage by H₂O₂. The reaction mixture for each of the last eight lanes contained 1 mm ATP (see separately bracketed lanes). (B) Stimulation of hTOP2α- and hTOP2β-mediated DNA cleavage by H₂O₂; 3 mm H₂O₂ was used in these reactions. (C) H₂O₂ does not stimulate TOP1-mediated DNA cleavage. (D) DNA double-strand breaks stimulated by H₂O₂ require active TOP2. The reactions were performed in three sets, one set without added TOP2, one set with added TOP2, and the other with heat-inactivated TOP2 (65°C for 15 min) (see three separately bracketed lanes). (E) DNA fragments induced by H₂O₂ are protein linked. After incubation of cleavage reactions, one set of reactions was terminated by treatment with 1% SDS in the absence of PK (another 60 min at 37°C) (see bracketed lanes under −PK). A duplicate set of reactions was terminated with 1% SDS plus 400 μg/ml PK and then incubation continued at 37°C for another 60 min (see bracketed lanes under +PK). The last half of the panel (last 13 lanes) were identical to the first half of panel (first 13 lanes), except that labeled DNA was first reacted with VM-26 or H₂O₂ in the absence of TOP2. Drug-treated DNA was then extracted with phenol and precipitated with ethanol, and resuspended in reaction mixtures containing calf thymus TOP2. All topoisomerase DNA cleavage assays were performed as described in Materials and Methods. Except for B, in which recombinant hTOP2α and hTOP2β were used, calf thymus topoisomerases were used in all cleavage assays. The concentrations of VM-26 (VM), camptothecin (CPT), and hydrogen peroxide (H₂O₂) were as indicated at the top of each lane.