Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites
Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites
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DOI：
10.1038/nprot.2008.54
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Kastan, Michael B.
中科院分区：
文献类型：
--
作者：
Berkovich, Elijahu;Monnat, Raymond J., Jr.;Kastan, Michael B.
DNA双链断裂（DSB）位点处蛋白质聚集体（病灶）的形成主要通过免疫染色进行研究，因此受限于光学显微镜的低分辨率和合适的选择性抗体的可用性。在这里，我们描述了一个系统，使用酶的创建位点特异性DNA DSB结合染色质免疫沉淀（ChIP），使分子探测的DSB在人类基因组内。在诱导I-PpoI酶和产生DSB后，细胞DNA和蛋白质交联并通过ChIP分析断裂位点的特异性蛋白质。该系统允许以高分辨率直接检测断裂部位的蛋白质和染色质动态，以及直接测量人类细胞中的DNA修复缺陷。从片段化的染色质开始，2-3 d即可获得结果。
The formation of protein aggregates ( foci) at sites of DNA double-strand breaks (DSBs) is mainly studied by immunostaining and is hence limited by the low resolution of light microscopy and the availability of appropriate and selective antibodies. Here, we describe a system using enzymatic creation of site-specific DNA DSBs within the human genome combined with chromatin immunoprecipitation (ChIP) that enables molecular probing of a DSB. Following induction of the I-PpoI enzyme and generation of DSBs, cellular DNA and proteins are crosslinked and analyzed by ChIP for specific proteins at the site of the break. The system allows the direct detection of protein and chromatin dynamics at the site of the break with high resolution, as well as direct measurement of DNA repair defects in human cells. Starting with fragmented chromatin, results can be achieved in 2-3 d.