NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation
NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation
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DOI：
10.15252/embj.2018101005
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发表时间：
2019-04-01
期刊：
影响因子：
11.4
通讯作者：
Cejka, Petr
中科院分区：
文献类型：
--
作者：
Anand, Roopesh;Jasrotia, Arti;Cejka, Petr
DNA 末端切除通过同源重组启动 DNA 双链断裂修复。 MRE11-RAD50-NBS1 和磷酸化 CtIP 通过 MRE11 催化的核酸内切 DNA 切割执行第一个切除步骤。与酿酒酵母中的同源物 Xrs2 相比，人类 NBS1 对这一过程至关重要，这凸显了高等真核生物中调节 MRE11 核酸酶的复杂机制。使用重构的系统，我们在此展示了 NBS1 通过其 FHA 和 BRCT 结构域，充当 CtIP 磷酸化的传感器。然后，NBS1 通过与 MRE11 的直接物理相互作用激活 MRE11-RAD50 核酸酶。在缺少 NBS1 的情况下，MRE11-RAD50 表现出较弱的核酸酶活性，需要 CtIP，但不严格要求其磷酸化。这确定了 CtIP 增强 MRE11 的至少两种机制：通过 NBS1 的磷酸化依赖性模式和不使用 NBS1 的磷酸化非依赖性模式。作为支持，我们证明在缺乏 NBS1 的 FHA 和 BRCT 结构域的情况下，体内会发生有限的 DNA 末端切除。总的来说，我们的数据表明，当 CtIP 广泛磷酸化时，NBS1 将 MRE11-RAD50 核酸酶限制在 S-G2 相。这定义了在 DNA 代谢中调节 MRE11 核酸酶的机制。
DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.