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Physico-Chemical Events

(PP004) Insight into the mechanisms leading to the production of double-strand breaks in supercoiled DNA by ¹²⁵I-Hoechst.

Balagurumoorthy, Pichumani^*,1, Chen, Kai¹, Bash, Ralph², Adelstein, S. James¹, Kassis, Amin¹, ¹ Department of Radiology, Boston, MA, USA² The Biodesign Institute, Tempe, AZ, USA

ABSTRACT- We have previously elucidated the kinetics of DNA strand breaks produced by low-energy Auger electrons after the decay of iodine-125 (¹²⁵I) positioned in the minor groove of DNA by the binding of ¹²⁵I-Hoechst 33342 (¹²⁵IH) [Kassis et al. Radiat Res 1999;151:167-76]. These experiments, carried out with supercoiled (SC) pUC19 plasmid DNA and a ¹²⁵IH:DNA molar ratio of 42:1, showed that approximately one double-strand break (DSB) was produced per decaying ¹²⁵I atom in both the presence and the absence of the hydroxyl radical scavenger DMSO (0.2 M). Recently, we hypothesized that this DSB yield might be an overestimate as a consequence of ¹²⁵IH decays occurring in nicked (N) DNA, i.e. in the remaining 41 DNA-bound ¹²⁵IH molecules. To test our hypothesis, experiments have been performed using SC pUC19 plasmid DNA and a ¹²⁵IH:DNA molar ratio of 3:1. Tritium-labeled SC pUC19 DNA was incubated with ¹²⁵IH (±0.2 M DMSO) at 4°C. Aliquots were removed from the incubation mixture at various time intervals (1–60 d), analyzed on 1% agarose gel, and the fractions of intact SC, N, and linear (L) DNA were quantified. Iodine-125 activity in the incubation mixtures was measured in a –counter at each time point. DNA samples from incubation mixtures were also imaged by atomic force microscopy (AFM). The data indicate that the solubility of ¹²⁵IH increased steeply during the first 6 days of incubation. Furthermore, and in agreement with our previous findings, (i) each decay of ¹²⁵I produces 3.01±0.19 single-strand breaks (SSB), and (ii) this SSB yield decreases to 0.33±0.02 in the presence of DMSO demonstrating that 90% of these breaks are mediated by hydroxyl radicals. The DSB yield per ¹²⁵IH decay in the absence as well as the presence of DMSO, while being equal (0.55±0.01 and 0.48±0.05, respectively), is about half of that determined previously (0.90±0.11). Visualization of N and L DNA by AFM confirms these observations. We conclude that (i) the higher DSB yield obtained previously was in fact a consequence of the decaying ¹²⁵IH in the nicked DNA molecules, and (ii) under our current conditions, the formation of L and N DNA, indicators of DSB and SSB, respectively, occurs exclusively from the decay of ¹²⁵IH in SC DNA. These findings accurately reflect the DSB yield produced by this minor-groove-bound Auger electron emitter.

Key words: Auger electron(s), iodine-125, double-strand break, minor groove binding of ¹²⁵I-Hoechst 33342