Balagurumoorthy, P., Chen, K., Bash, R. C., Adelstein, S. J. and Kassis, A. I. Mechanisms Underlying Production of Double-Strand Breaks in Plasmid DNA after Decay of 125I-Hoechst. Radiat. Res. 166, 333–344 (2006).
Previously, the kinetics of strand break production by 125I-labeled m-iodo-p-ethoxyHoechst 33342 (125IEH) in supercoiled (SC) plasmid DNA had demonstrated that ∼1 DSB is produced per 125I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an 125IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 125IEH molecules still bound to nicked (N) DNA. To test our hypothesis, 125IEH was incubated with SC pUC19 plasmids (125IEH:DNA ratio of ∼3:1) and the SSB and DSB yields were quantified after the decay of 125I. As predicted, the number of DSBs produced per 125I decay is one-half that reported previously (∼0.5 compared to ∼1, ± DMSO) whereas the number of SSBs (∼3/125I decay) is similar to that obtained previously (∼90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after 125IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of 125IEH bound to DNA, the DSB yield can be augmented erroneously by 125I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an 125IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.