SINGLE-CELL GEL-ELECTROPHORESIS APPLIED TO THE ANALYSIS OF UV-C DAMAGE AND ITS REPAIR IN HUMAN-CELLS
SINGLE-CELL GEL-ELECTROPHORESIS APPLIED TO THE ANALYSIS OF UV-C DAMAGE AND ITS REPAIR IN HUMAN-CELLS
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DOI：
10.1080/09553009214552161
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发表时间：
1992-09-01
影响因子：
2.6
通讯作者：
COLLINS, AR
中科院分区：
文献类型：
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作者：
GEDIK, CM;EWEN, SWB;COLLINS, AR
真核细胞中的 DNA 断裂可以通过包埋在琼脂糖中的细胞的碱性电泳来检测。含有断裂的 DNA 沿阳极方向延伸，形成类似彗星尾巴的图像。我们采用了单细胞凝胶电泳 (SCGE) 程序，使用 HeLa 细胞研究 UV-C 辐射诱导的 DNA 损伤和修复。 UV-C 本身不会引起 DNA 断裂，尽管 UV-C 损伤的细胞修复会产生 DNA 断裂作为中间体，但这些断裂的寿命太短，无法被 SCGE 检测到。将 UV-C 照射的细胞与 DNA 合成抑制剂 aphidicolin 一起孵育，即使在剂量低至 0.5 J m-2 且孵育时间短至 5 分钟后，也会导致不完全修复位点的积累达到 SCGE 易于检测到的水平。我们还使用 SCGE 研究了通透性细胞中 UV-C 依赖性切割、修复合成和连接。最后，我们在 UV-C 照射后与外源性 UV 核酸内切酶一起孵育可渗透细胞，通过 SCGE 和碱性解旋检查随后的断裂，以便以 DNA 断裂频率表达电泳方法的结果。因此可以估计 SCGE 技术的灵敏度；每 10(9) 道尔顿仅检测到 0.1 个 DNA 断裂。
DNA breaks in eukaryotic cells can be detected by alkaline electrophoresis of cells embedded in agarose. DNA containing breaks extends in the direction of the anode forming an image resembling the tail of a comet. We have adapted this procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m-2 and incubation for as little as 5 min. We have also used SCGE to study UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, we have incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examining the consequent breaks both by SCGE and by alkaline unwinding in order to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10(9) daltons are detected.