Immunoreactivity to antinucleoside antibodies in X-irradiated HeLa cells

Synchronized HeLa cells were stained with antibodies to purine and pyrimidine nucleosides by immunofluorescent and immunoperoxidase techniques. These antibodies react only with denatured or single-stranded regions of DNA. Nuclear attachment of antibody was seen only during the period of DNA synthesis as determined by ³H-thymidine incorporation. Positive nuclear immunoreactivity was seen in approx. 15% of cells obtained by mitotic selection at a time corresponding to the G 1 phase. After exposure to ionizing radiation, 80% of the G 1 cells were reactive. Induction of immunoreactivity was dose dependent over the range of 100 to 1 000 rads. Treatment of irradiated G 1 cells with deoxyribonuclease completely eliminated the positive nuclear reaction. Exposure to ribonuclease had no effect. Incubation of the G 1 cells for 90 min at 37 or 0–4 °C after the administration of 1 000 rads resulted in a prompt decrease of immunoreactivity to control levels. However, in the presence of 0.04 μg/ml actinomycin D, positive nuclear staining remained at high levels. No such effect could be observed as a result of exposure to cytosine arabinoside or hydroxyurea. It is concluded that X-irradiation of G 1 HeLa cells produces single-stranded regions in nuclear DNA that can be detected by anti-pyrimidine and anti-purine antibodies.