Auger electron-induced double-strand breaks depend on DNA topology

Abstract

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.

FIG. 1

Dissolved ¹²⁵I activity present in incubations of supercoiled (SC, ■), linear (L, ●) and nicked (N, ▲) forms of ³HT-pUC19 DNA with ¹²⁵IEH as a function of time.

FIG. 2

Agarose gel analysis of supercoiled (SC), nicked (N) and linear (L) forms of ³HT-pUC19 plasmid DNA incubated with ¹²⁵IEH at 4°C in PBS (pH 7.4): lanes C_SC, C_N and C_L represent control incubations of supercoiled, nicked and linear DNA without ¹²⁵IEH; − and + indicate absence and presence of DMSO (0.2 M), respectively. Gels containing ethidium bromide were visualized using ultraviolet (320 nm) transillumination.

FIG. 3

Quantitative analysis of agarose gel electrophoresis for disappearance of linear ³HT-pUC19 plasmid DNA (panel A) and nicked ³HT-pUC19 plasmid DNA (panel B) as a function of ¹²⁵I decays accumulated in the absence (■) and presence (□) of DMSO.

FIG. 4

Fluorescence titration curves of supercoiled (SC), linear (L) and nicked (N) forms of pUC19 DNA with Hoechst 33342 (0.169 μM) in PBS (pH 7.4). Fluorescence maxima for all three forms of pUC19 DNA occur at approximately the same DNA concentration (20 μM), indicating saturation of ligand binding to DNA. In ¹²⁵IEH–DNA incubations, the concentration of ¹²⁵IEH is 284 μM, ensuring that there is little unbound ¹²⁵IEH in mixtures.

FIG. 5

Mean DSB yield per DNA molecule for supercoiled (SC) pUC19 plasmid DNA without or with DMSO, calculated from the appearance of linear (L) DNA as a function of accumulated ¹²⁵I decays. Linear regressions were carried out and R² was obtained using Origin. Regression lines were forced through zero.

FIG. 6

Mean DSB yield per DNA molecule for linear and nicked DNA: linear form −DMSO (panel A); linear form +DMSO (panel B); nicked form −DMSO (panel C); nicked form +DMSO (panel D). DSB yields were calculated from the rate of disappearance of intact linear or nicked DNA as a function of accumulated ¹²⁵I decays. Linear regressions were carried out and R² was obtained using Origin. Regression lines were forced through zero.

FIG. 7

Proposed mechanisms for DSB formation in pUC19 DNA caused by ¹²⁵I decays from minor-groove-bound ¹²⁵IEH. Supercoiled DNA (panel A), linear DNA (panel B), nicked DNA (panel C).