Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System
Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System
复制标题
使用旋转凝胶电泳系统通过脉冲场凝胶电泳检测大肠杆菌中博莱霉素诱导的 DNA 双链断裂
DOI：
10.1007/978-1-0716-0323-9_14
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Hanada Katsuhiro
中科院分区：
文献类型：
--
作者：
Inoue Naomi;Narahara Hisashi;Nishida Yoshihiro;Hanada Katsuhiro
DNA双链断裂（DSB）是最具遗传毒性的病变之一，未修复的DSB会导致染色体不稳定并最终导致细胞死亡。定量标记，例如哺乳动物细胞中的磷酸化组蛋白 H2AX (γ-H2AX) 和 p53 结合蛋白 1 (53BP1) 焦点，无法用于检测原核生物中的 DSB。因此，作为一种替代方法，脉冲场凝胶电泳 (PFGE) 被广泛用于通过将断裂的 DNA 分子与完整的 DNA 分离来分析它们。在这里，我们使用旋转凝胶电泳 (RGE) 系统通过 PFGE 检查了博来霉素 (BLM) 诱导的 DSB 的积累。我们定义了两组具有明显优势的参数；第一个侧重于分析断裂 DNA 片段的大小，而第二个则允许直接比较菌株和处理之间 DSB 的积累。该方法是研究基因组完整性和遗传毒性物质表征的强大工具。
DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal instability and eventually cause cell death. Quantitative markers, such as phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1) foci in mammalian cells, are not available for the detection of DSBs in prokaryotes. Therefore, as an alternative method, pulsed-field gel electrophoresis (PFGE) is widely used to analyze broken DNA molecules by separating them from intact DNA. Here, we examined the accumulation of bleomycin (BLM)-induced DSBs by PFGE, using a rotating gel electrophoresis (RGE) system. We defined two sets of parameters with distinct advantages; the first one focuses on the analysis of the size of the broken DNA fragments, whereas the second allows for the direct comparison of the accumulation of DSBs among strains and treatments. This method represents a powerful tool for the study of genomic integrity and the characterization of genotoxic substances.