DAMAGE AT 2 LEVELS OF DNA FOLDING MEASURED BY FLUORESCENT HALO TECHNIQUE IN X-IRRADIATED L5178Y-R AND L5178Y-S CELLS .2. REPAIR

In the preceding paper we described the properties of nucleoids analyz ed with the fluorescent halo assay at pH 6.9 and 9, as well as in the presence of reducing and chelating agents and after X-irradiation. We found analogies between the properties of type I and II nucleoids, as examined by Lebkowski and Laemmli (1982), and nucleoids analyzed with the fluorescent halo assay. We concluded that radiation-inflicted dama ge at two levels of DNA folding is measured at pH 6.9 and 9. In this p aper we examined repair of damage to the nucleoid structure as assayed by the fluorescent halo method in X-irradiated L5178Y (LY) sublines; R (radiation resistant, D-0=1.4 Gy) and S (radiation sensitive, D-0=0. 5 Gy). Halo diameters were measured after cell lysis in the presence o f propidium iodide (PI; 0.5 to 50 mu g/ml) at pH 6.9 and 9. The abilit y of DNA to be rewound at 10-50 mu g/ml of PI was impaired by X-irradi ation and partly restored during 90-min post-irradiation incubation, i ndicating damage to the superhelical structure and its partial restora tion. The exponential time constants for repair were 10.1 min (LY-S, 6 Gy), 11.2 min (LY-R, 12 Gy), and 20.3 min (LY-s, 12 Gy) when measured at pH 9. In X-irradiated (12 Gy) LY-S cells, slower restoration of DN A supercoiling was observed at pH 9 than at pH 6.9. The presence of la bile lesions at pH 9 did not prevent restoration of the higher-order D NA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.