Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks.
Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks.
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发表时间：
2001-05
期刊：
影响因子：
11.2
通讯作者：
K. Rothkamm;M. Kühne;P. Jeggo;M. Löbrich
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文献类型：
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作者：
K. Rothkamm;M. Kühne;P. Jeggo;M. Löbrich
在哺乳动物细胞中，DNA双链断裂（DSB）的修复主要有两种途径：非同源末端连接（NHEJ）和同源重组（HR）。已知NHEJ的失活导致与肿瘤发生风险增加相关的自发和辐射诱导的染色体重排水平升高。这就提出了让国家高等法院发挥看守作用的想法。然而，目前尚不清楚NHEJ本身是否也会引起重排。为了研究，在DNA水平上，在NHEJ的缺陷对基因组重排的形成的影响，我们应用了基于Southern杂交的测定，其允许鉴定和定量电离辐射产生的不正确重新连接的DSB末端。在高剂量率（23戈伊/min）的80戈伊X射线照射后，野生型细胞在24小时内通过不正确的重新连接修复了50%的诱导的DSB。在NHEJ缺陷型细胞中，DSB错误连接的频率显著降低。低剂量率的实验中，细胞暴露于80戈伊超过14天的修复条件下，导致没有检测到的错误重新连接在野生型细胞，但揭示了10%的错误重新连接的频率在NHEJ缺陷细胞。这表明在分离断裂的情况下，NHEJ缺陷导致基因组重排，与染色体研究一致。然而，如果多个DSB重合，即使是野生型细胞也经常形成基因组重排。这些修复事件在Ku80-、DNA-PKcs-和DNA连接酶IV-缺陷细胞中不存在，但在RAD 54（-/-）细胞中存在。这强烈地表明，NHEJ除了其看护作用之外，还具有影响基因组重排的潜力。我们建议，它作为一个有效的途径，重新连接正确的中断结束的情况下，分开的休息，但产生基因组重排，如果DSB是在时间和空间上接近。
Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.