Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. Pt. 1

We examined, by the fluorescent halo assay, alterations in the nucleoid structure (structure formed from cells under mild lysis conditions: In non-ionic detergent Triton X-100, 0.0005% and 1.5 mol/l NaCl) of L5178Y (LY) cell sublines which had been untreated, treated with reducing/chelating agents (β-mercaptoethanol or sodium diethyl dithiocarbamate (DDTC(Na)) or X-irradiated. These sublines differ in radiation sensitivity: LY-R is more resistant (D0=1.1 Gy) and LY-S more sensitive (D0=0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI) (0.5 to 50 μg/ml) at pH 6.9 or 9. The maximal DNA unwinding in PI was obtained at 7.5 μg/ml PI, at both pH 6.9 and 9 in both sublines; the maximal halo diameter was larger in LY-S than in LY-R cells. In nucleoids from both sublines DNA could be rewound at higher (10-50 μg/ml) PI concentrations both at pH 6.9 and 9. This ability was impaired by mercaptoethanol or DDTC(Na) (at pH 9) or by X-irradiation, indicating damage and/or alteration in the DNA superhelical structure. The susceptibility to reducing/chelating agents was greater in LY-S than in LY-R nucleoids, pointing to differences in chromatin structure between these sublines. The amount of X-ray inflicted damage was higher, when measured at pH 9 than at pH 6.9 and was about twice larger in LY-S than in LY-R nucleoids, when the cells were irradiated with the same X-ray dose. From analogies between the behaviour of nucleoids under the above-described conditions and nucleoid type I and II sedimentation, as examined by Lebkowski and Laemmli (1982) we conclude that damage at two levels of DNA folding is measured at pH 6.9 and 9. (orig.)