The Shape of DNA Elution Dose-response Curves Under Non-denaturing Conditions: The Contribution of the Degree of Chromatin Condensation

Abstract

We have re-examined the effect of detergent type, pH and temperature of lysis on the shape of the DNA elution dose response under non-denaturing conditions using plateau-phase CHO cells. Results practically identical to those previously published (Okayasu and Iliakis, 1989) were obtained, with a 1 h incubation at 60°C during lysis with sodium-N-laurylsarcosine (NLS) resulting in almost linear dose-response curves. We also examined chromatin decondensation as a contributing factor in the linearization observed in the elution dose-response curve under the above conditions. When nuclei with condensed chromatin were prepared from irradiated cells, applied on the filter and lysed with NLS at room temperature, a shoulder-type elution dose-response curve was obtained only slightly higher than that of cells lysed under the same conditions. However, when nuclei prepared from irradiated cells were applied on the filter after relaxation of chromatin by incubation in low ionic strength buffer and lysed with NLS at room temperature, an almost linear dose-response curve was obtained similar to that of cells lysed with NLS at 60°C. Lysis with NLS at 60°C of nuclei with relaxed chromatin did not further modify the DNA elution dose-response curve. Based on these results we propose that the linearization of the DNA elution dose-response curve observed after chromatin decondensation reflects a reduction in the degree of chromatin compactness in the nuclear complexes that leads to a relatively uniform distribution of the DNA on the filter and reduces trapping of elutable material in the compact nuclear structures otherwise present. Since high radiation doses dissolve compact nuclear structures, trapping of elutable material is expected to be highest at low doses of radiation, leading to the observed reduction in the fraction of DNA eluted per Gy at low versus high radiation doses and thus to the observed shoulder. Furthermore, we propose that the linearization of the DNA elution dose-response curves observed in cells lysed in NLS at 60°C may also be due to a decondensation of the nuclear complexes on the filter as a result of the combined action of detergent and high temperature. The notion of a correlation between DNA elution dose response and cell radiosensitivity was examined in two human (SQ20B, SCC61) and two Chinese hamster (V-79, irs-2) cell lines with widely different radiosensitivities. We found that radiation-sensitive and radiation-resistant cell lines give similar DNA elution dose-response curves, suggesting that elution dose response and cell radiosensitivity are not necessarily coupled.