From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by (125)I, targeted to plasmid DNA by m-[(125)I]iodo-p-ethoxyHoechst 33342 ((125)IEH). DSB yields per (125)I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74+/-0.11 and 1.62+/-0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52+/-0.02), despite the same affinity of all forms for (125)IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05+/-0.07 and 0.76+/-0.03 per (125)I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.