Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses
Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses
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DOI：
10.1074/jbc.m112.384750
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发表时间：
2012-09-14
影响因子：
4.8
通讯作者：
Wu, Xiaohua
中科院分区：
文献类型：
--
作者：
He, Jing;Shi, Linda Z.;Wu, Xiaohua
Mre11-Rad50-Nbs1(MRN)复合体在检查点激活和双链断裂(DSB)修复中起关键作用。Rad50锌钩结构域介导了依赖锌的MRN的复合体间关联，这对DNA拴系是重要的。在酵母中的研究表明，Rad50锌钩结构域是MRN功能所必需的，但它在哺乳动物细胞中的作用尚不清楚。我们发现，人类Rad50钩突变体在多种DNA损伤反应中存在严重缺陷，包括ATM(共济失调毛细血管扩张突变)激活、同源重组、对IR的敏感性以及ATR途径的激活。通过活细胞成像，我们观察到Rad50钩突变体未能被招募到染色体DSB上，这表明了一种新的机制，导致了Rad50钩突变体观察到的严重缺陷。体外分析表明，锌离子能促进野生型MR结合双链DNA，而不能促进钩状突变体结合双链DNA。在体内，Rad50挂钩突变体在H2 AX熟练和缺陷细胞中都不能被招募到染色体DSB，这表明Rad50挂钩突变体在与染色体DSB末端的直接结合方面受到损害。我们认为Rad50锌钩结构域对于MRN与DSB的初始结合是重要的，导致ATM激活以磷酸化H2AX，从而将更多的MRN招募到DSB两侧的染色体区域。我们的研究揭示了Rad50锌挂钩结构域在建立和维持MRN招募到染色体DSB中的关键作用，并提出了Rad50锌挂钩结构域如何促进DNA修复和检查点激活的重要机制。
The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn2+ promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.