Kapiszewska M, Szumiel I, Lange C S
Institute of Molecular Biology, Jagiellonian University, Kraków, Poland.
Radiat Environ Biophys. 1994;33(1):35-44. doi: 10.1007/BF01255272.
In the preceding paper we described the properties of nucleoids analyzed 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 damage at two levels of DNA folding is measured at pH 6.9 and 9. In this paper 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, D0 = 1.4 Gy) and S (radiation sensitive, D0 = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI; 0.5 to 50 micrograms/ml) at pH 6.9 and 9. The ability of DNA to be rewound at 10-50 micrograms/ml of PI was impaired by X-irradiation and partly restored during 90-min post-irradiation incubation, indicating damage to the superhelical structure and its partial restoration. 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 DNA supercoiling was observed at pH9 than at pH 6.9. The presence of labile lesions at pH 9 did not prevent restoration of the higher-order DNA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.
在之前的论文中，我们描述了通过荧光晕圈测定法在pH 6.9和9的条件下，以及在存在还原剂和螯合剂的情况下和X射线照射后对类核体性质的分析。我们发现，Lebkowski和Laemmli（1982年）所检测的I型和II型类核体的性质与通过荧光晕圈测定法分析的类核体之间存在相似之处。我们得出结论，在pH 6.9和9的条件下，可以检测到DNA折叠两个水平上的辐射损伤。在本文中，我们通过荧光晕圈法检测了X射线照射的L5178Y（LY）亚系（R系，辐射抗性，D0 = 1.4 Gy；S系，辐射敏感性，D0 = 0.5 Gy）中类核体结构损伤的修复情况。在pH 6.9和9的条件下，于碘化丙啶（PI；0.5至50微克/毫升）存在的情况下进行细胞裂解后，测量晕圈直径。X射线照射会损害DNA在10 - 50微克/毫升PI存在下重新缠绕的能力，并且在照射后90分钟的孵育过程中部分恢复，这表明超螺旋结构受到损伤并部分恢复。在pH 9条件下测量时，修复的指数时间常数分别为10.1分钟（LY - S，6 Gy）、11.2分钟（LY - R，12 Gy）和20.3分钟（LY - s，12 Gy）。在X射线照射（12 Gy）的LY - S细胞中，在pH 9条件下观察到DNA超螺旋的恢复比在pH 6.9条件下更慢。根据LY - S细胞在pH 6.9条件下的DNA重新缠绕情况估计，在pH 9条件下不稳定损伤的存在并不妨碍高阶DNA结构的恢复。
