No detectable misrejoining in double-minute chromosomes

Pulsed-field gel electrophoresis combined with Southern hybridization and r are-cutting restriction endonuclease digestion has been used recently to qu antify misrejoining of DNA double-strand breaks (DSBs) resulting from expos ure to ionizing radiation. Measurements are made 24 h after a high dose of radiation. These studies have suggested that a large fraction of DSBs are m isrejoined to result in gross rearrangements. In the experiments described here, we show that elimination of broken DNA also eliminates "misrejoined" DNA. Mouse cells resistant to high levels of methotrexate by virtue of 100- fold amplification of the dyhydrofolate reductase (Dhfr) gene were treated with 50 and 100 Gy of ionizing radiation. The cells were allowed to repair the damage for 24 h. After the repair period, the cells were immobilized in agarose. Aliquots of each sample were pre-electrophoresed to remove linear DNA molecules smaller than 6 Mbp resulting from apoptosis or necrosis. The samples repairing damage from 50 or 100 Gy that did not receive the pre-el ectrophoresis showed high levels of label in a region of the lane that coul d be due to misrejoining DNA molecules. However, when the DNA from cells un dergoing apoptosis or necrosis was removed from these samples, the levels o f "misrejoined" DNA were reduced to levels far below those of unirradiated controls. These results suggest that other radiation-induced effects presen t 24 h after irradiation with 50 or 100 Gy are more significant than misrej oining for altering hybridization to regions of the lane outside the specif ic bands. Measurements of misrejoining using PFGE, rare-cutting restriction endonucleases, and Southern hybridization are likely to be compromised by nonspecific hybridization to broken and difficult-to-digest DNA resulting f rom apoptosis or necrosis. (C) 1999 by Radiation Research Society.