Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre
Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre
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DOI：
10.1038/ncb997
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发表时间：
2003-06-01
影响因子：
21.3
通讯作者：
Rothstein, R
中科院分区：
文献类型：
--
作者：
Lisby, M;Mortensen, UH;Rothstein, R
DNA双链断裂修复（DSBR）是维持所有生物体基因组完整性的重要过程。为了在细胞水平上研究这一过程，我们设计了一个荧光标记的DNA双链断裂（DSB）系统，在酵母酿酒酵母中可视化单细胞中的体内DSBR。使用这个系统，我们首次证明，Rad52 DNA修复灶和DSB共定位。延时显微镜显示，Rad52蛋白的重新定位成焦点组装是一个快速和可逆的过程。此外，对DNA损伤检查点缺陷细胞的分析为DNA修复和随后从检查点停滞中释放之间的协调提供了直接证据。最后，对经历多个DSB的细胞的分析表明，Rad52病灶是能够同时招募一个以上DSB的DNA修复中心。
DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.