Characterization of complex apurinic/apyrimidinic-site clustering associated with an authentic site-specific...

Radiation lethality is largely attributed to radiation-induced DNA double-strand breaks (DSBs). A range of structural complexity is predicted for radiation-induced DSBs. However, this lesion has never been analyzed in isolation at the molecular level. To address this problem, we have created authentic site-specific radiation-induced DSBs in plasmid DNA by triplex-forming oligonucleotide-targeted 125I decay. No significant difference in DSB yield was observed after irradiation in the presence or absence of the radical scavenger DMSO, suggesting that DSB formation is a result of the direct effect of the radiation. A restriction fragment terminated by the DSB was isolated and probed with the Escherichia coli DNA repair enzyme endonuclease IV (endo IV), which recognizes apurinic/apyrimidinic (AP) sites. Enzymatic probing demonstrated clustering of AP sites within 10 bases of the 125I-targeted base in the DNA duplex. Our results suggest scavengeable radicals may not play a large role in the generation of AP sites associated with DSB formation, because at least 30% of all fragments have endo IV-sensitive sites, regardless of irradiation conditions. An internal control fragment recovered from the 125I linearized plasmid did not exhibit endo IV sensitivity in excess of that observed for a similar fragment recovered from an undamaged plasmid. Thus, AP site clustering proximal to the DSB resulted from the 125I decays responsible for DSB formation and was not due to untargeted background irradiation.