Multiply damaged sites (MDSs) consist of two or more damages within 20 base pairs (bps) and are introduced into DNA by ionizing radiation. Using a plasmid assay, we previously demonstrated that repair in Escherichia coli generated a double strand break (DSB) from two closely opposed uracils when uracil DNA glycosylase initiated repair. To identify the enzymes that converted the resulting apurinic/apyrimidinic (AP) sites to DSBs, repair was examined in bacteria deficient in AP site cleavage. Since exonuclease III (xth) and endonuclease IV (nfo) mutant bacteria were able to introduce DSBs at the MDSs, we generated unique bacterial mutants deficient in UvrA, Xth and Nfo. However, the additional disruption of nucleotide excision repair (NER) did not prevent DSB formation. xth- nfo- nfi- bacteria also converted the MDSs to DSBs, ruling out endonuclease V as the candidate AP endonuclease. By using MDSs containing tetrahydrofuran (an AP site analog), it was determined that even in the absence of Xth, Nfo, NER and AP lyase cleavage, DSBs were formed from closely opposed AP sites. This finding implies that there is an unknown enzyme/repair pathway for MDSs, and multiple underlying repair systems in cells that can process closely opposed DNA damage into lethal lesions following exposure to ionizing radiation.