Multiply damaged sites (MDS) are defined as greater than/equal to two lesions within 10-15 bp and are generated in DNA by ionizing radiation. In vitro repair of closely opposed base damages > or =2 bp apart results in a double strand break (DSB). This work extends the in vitro studies by utilizing clusters of uracil DNA damage as model lesions to determine whether MDS are converted to DSBs in bacteria. Lesions were positioned within the firefly luciferase coding region, transformed into bacteria (wild-type, uracil DNA glycosylase-deficient, ung-, or exonuclease III and endonuclease IV-deficient, xth-nfo-) and luciferase activity measured following repair. DSB formation was expected to decrease activity. Two closely opposed uracils separated by < or =7 bp decreased luciferase activity in wild-type and xth-nfo-, but not ung- bacteria. Growth of bacteria to obtain plasmid-containing colonies demonstrated that the plasmid was destroyed following the mis-repair of two uracils positioned 7 bp apart. This study indicates a DSB is formed when uracil DNA glycosylase initiates repair of two closely opposed uracils < or =7 bp apart, even in the absence of the major apurinic endonucleases. This work supports the in vitro studies and demonstrates that DNA repair is not always advantageous to cells.