Spontaneous DNA breakage in single living Escherichia coli cells
Spontaneous DNA breakage in single living Escherichia coli cells
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DOI：
10.1038/ng2051
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发表时间：
2007-06-01
期刊：
影响因子：
30.8
通讯作者：
Rosenberg, Susan M.
中科院分区：
文献类型：
--
作者：
Pennington, Jeanine M.;Rosenberg, Susan M.
自发性DNA断裂被预测为DNA复制的频繁、不可避免的结果，并且被认为是导致癌症和进化的大部分基因组变化的基础（1-3）。尽管其重要性，一直很少有直接测量的数量，类型，来源和命运的自发DNA损伤活细胞。我们提出了一个直接的，敏感的流式细胞术检测在单个活的大肠杆菌细胞的DNA损伤能够诱导SOS DNA损伤反应，我们报告其使用的定量自发DNA双链断裂（DSB）。我们报告了单染色体DSB的有效检测，自发断裂率比预期低20- 100倍。此外，我们牵连的DNA复制在自发DSB的起源与发现较少的自发DSB的突变体与改变DNA聚合酶III。这些数据表明，自发DSB诱导的基因组变化和不稳定性比以前认识到的强20-100倍。最后，流式细胞仪显示两个主要的细胞命运后，自发DNA损伤：活力与或不恢复增殖。
Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution(1-3). Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage similar to 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.