使用旋转凝胶电泳系统通过脉冲场凝胶电泳检测博来霉素诱导的大肠杆菌DNA双链断裂。

Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System.

作者信息

Inoue Naomi, Narahara Hisashi, Nishida Yoshihiro, Hanada Katsuhiro

机构信息

Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Oita, Japan.

Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Oita, Japan.

出版信息

Methods Mol Biol. 2020;2119:155-163. doi: 10.1007/978-1-0716-0323-9_14.

DOI:10.1007/978-1-0716-0323-9_14

PMID:31989523

Abstract

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.

摘要

DNA双链断裂（DSB）是最具基因毒性的损伤之一，未修复的DSB可导致染色体不稳定并最终导致细胞死亡。在原核生物中，尚无用于检测DSB的定量标志物，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。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检测博来霉素（BLM）诱导的DSB的积累。我们定义了两组具有不同优势的参数；第一组侧重于分析断裂DNA片段的大小，而第二组允许直接比较不同菌株和处理之间DSB的积累情况。该方法是研究基因组完整性和基因毒性物质特征的有力工具。