Detection of DNA double-strand breaks by pulsed-field gel electrophoresis
Detection of DNA double-strand breaks by pulsed-field gel electrophoresis
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DOI：
10.1111/gtc.12457
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发表时间：
2017-01-01
期刊：
影响因子：
2.1
通讯作者：
Hanada, Katsuhiro
中科院分区：
文献类型：
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作者：
Kawashima, Yuri;Yamaguchi, Nahomi;Hanada, Katsuhiro
DNA双链断裂（DSB）是最具细胞毒性的DNA损伤之一，因为未修复的DSB会导致染色体畸变和细胞死亡。虽然许多生理性DSB发生在DNA复制位点，但其分子机制仍然知之甚少。因此，需要开发一种高度特异性的方法来检测含有DNA复制位点的DSB片段。在这里，我们调查是否脉冲场凝胶电泳（PFGE）结合可视化的DNA复制位点的免疫印迹使用卤化脱氧尿苷，如BrdU和IdU，是足够的检测。我们的方法使我们能够重现以前报告的数据。此外，该方法也被应用于检测细菌感染诱导的人染色体DNA上的DSB。基于我们的研究结果，我们建议，这种策略结合PFGE与免疫印迹分析将适用于研究分析的机制细节的DNA修复，DNA损伤反应和DNA损伤剂的活性。
A DNA double-strand break (DSB) is one of the most cytotoxic DNA lesions because unrepaired DSBs cause chromosomal aberrations and cell death. Although many physiological DSBs occur at DNA replication sites, the molecular mechanisms underlying this remain poorly understood. There was therefore a need to develop a highly specific method to detect DSB fragments containing DNA replication sites. Here we investigated whether pulsed-field gel electrophoresis (PFGE) combined with visualization of DNA replication sites by immunoblotting using halogenized deoxyuridines, such as BrdU and IdU, was sufficient for this detection. Our methodology enabled us to reproduce previously reported data. In addition, this methodology was also applied to the detection of bacterial infection-induced DSBs on human chromosomal DNA. Based on our findings, we propose that this strategy combining PFGE with immunoblot analysis will be applicable to studies analyzing the mechanistic details of DNA repair, the DNA damage response and the activity of DNA-damaging agents.