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Author:
Date:
2013-09-20
[Abstract] Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, finally, process and ligate the ends.
Briefly, NHEJ starts with Ku protein. Ku binds the broken DNA ends and recruits the catalytic subunit of DNA dependent protein kinase (DNA-PKcs) forming DNA-PK. After processing, the XRCC4/Ligase IV complex executes the final ligation stimulated by Cernunnos-XLF.
Here, we describe an end-synapsis assay. This assay can be used in order to delineate which proteins are necessary to bring the ...
[摘要] 许多环境因素诱导DNA中的双链断裂（DSB）。未修复或不正确修复的DSB可导致细胞死亡或癌症。非同源末端连接是真核生物中的主要DNA双链断裂修复途径。在NHEJ途径期间，几种蛋白质识别和结合DNA末端，将末端引入突触复合物，最后进行处理并连接末端。
简言之，NHEJ起始于Ku蛋白。 Ku结合断裂的DNA末端，并招募形成DNA-PK的DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基。处理后，XRCC4 /连接酶IV复合物执行Cernunnos-XLF刺激的最终连接。
在这里，我们描述一种末端突触检测。可以使用该测定法来描绘在NHEJ期间将DNA末端置于稳定突触复合物中所必需的蛋白质。简言之，来自人细胞的NHEJ感受态提取物与结合链霉抗生物素蛋白包被的磁珠和相同的可溶性放射性标记片段的双链DNA片段一起温育。然后将微珠在温和的盐缓冲液中洗涤，并通过闪烁计数测量用珠回收的放射性。没有提取物或无DNA珠的对照实验平行进行以确定非特异性背景。
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