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(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
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