Characterization of a complex 125I-induced DNA double-strand break: implications for repair

To examine the role of radiation-induced DNA double-strand break (DSB) structural organization in DSB repair, and characterize the structural features of 125I-induced DSBs that may impact the repair process.

Plasmid DNA was linearized by sequence-specific targeting using an 125I-labeled triplex-forming oligonucleotide (TFO). Following isolation from agarose gels, base damage structures associated with the DSB ends in plasmids linearized by the 125I-TFO were characterized by probing with the E. coli DNA damage-specific endonuclease and DNA-glycosylases, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-glycosylase (Fpg).

Plasmid DNA containing DSBs produced by the high-LET-like effects of 125I-TFO has been shown to support at least 2-fold lower end joining than gamma-ray linearized plasmid, and this may be a consequence of the highly complex structure expected near an 125I-induced DSB end. Therefore, to determine if a high density of base damage exists proximal to the DSBs produced by 125I-TFOs, short fragments of DNA recovered from the DSB end of 125I-TFO-linearized plasmid were enzymatically probed. Base damage and AP site clustering was demonstrated within 3 bases downstream and 7 bases upstream of the targeted base. Furthermore, the pattern and extent of base damage varied depending upon the presence or absence of 2 M DMSO during irradiation.

125I-TFO-induced DSBs exhibit a high degree of base damage clustering proximal to the DSB end. At least 60% of the nucleotides within 10 bp of the 125I decay site are sensitive to cleavage by endo IV, endo III, or Fpg following damage accumulation in the presence of DMSO, whereas > or = 80% are sensitive in the absence of DMSO. The high degree of base damage clustering associated with the 125I-TFO-induced DSB end may be a major factor leading to its negligible in vitro repair by the non-homologous end-joining pathway (NHEJ).