Differences in the DNA supercoiling response of irradiated cell lines from ataxia-telangiectasia versus unaffected individuals

In this study the manifestation of DNA damage at the nucleoid level was examined in several AT cell lines using an image analysis system to directly visualize and measure the changes in DNA loop size which occur when increasing concentrations of propidium iodide (PI) are used to titrate the DNA supercoiling response (the 'fluorescent halo assay'). This response consists of a relaxation (0.5-7.5 micrograms/ml PI) and rewinding phase (10-50 micrograms/ml PI), the latter of which is impaired by the presence of DNA strand breaks in irradiated cells. In addition to the inhibition of DNA rewinding seen immediately after irradiation at 0 degrees C, the supercoiling response of AT diploid fibroblasts indicated an increased amount of DNA unwinding compared to fibroblasts from unaffected individuals. This difference appeared to saturate, since the excess in DNA loop size over that seen in irradiated fibroblasts from unaffected individuals remained constant after 5 Gy. These results may reflect a greater instability of the DNA-nuclear matrix attachment points in irradiated AT fibroblasts. The DNA supercoiling response in irradiated transformed AT fibroblasts and AT lymphoblasts did not differ from that observed in unaffected cells of the same type. However, all of the immortalized cell lines (AT and unaffected) had inherently larger DNA loop sizes than diploid fibroblasts and exhibited excess unwinding after irradiation.