Mechanisms underlying production of double-strand breaks in plasmid DNA after decay of 125I-Hoechst

Previously, the kinetics of strand break production by I-125-labeled m-iodo-p-ethoxyHoechst 33342 ((IEH)-I-125) in supercoiled (SC) plasmid DNA had demonstrated that similar to 1 DSB is produced per I-125 decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (IEH)-I-125: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 (IEH)-I-125 molecules still bound to nicked (N) DNA. To test our hypothesis, (IEH)-I-125 was incubated with SC pUC19 plasmids ((IEH)-I-125:DNA ratio of similar to 3:1) and the SSB and DSB yields were quantified after the decay of I-125. As predicted, the number of DSBs produced per I-125 decay is one-half that reported previously (similar to 0.5 compared to similar to 1, +/- DMSO) whereas the number of SSBs (similar to 3/I-125 decay) is similar to that obtained previously (similar to 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (IEH)-I-125 decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (IEH)-I-125 bound to DNA, the DSB yield can be augmented erroneously by 125 1 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 (IEH)-I-125 molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules. (c) 2006 by Radiation Research Society