Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland.
Department of Gynecology, University of Zurich, Schlieren, Switzerland.
EMBO J. 2019 Apr 1;38(7). doi: 10.15252/embj.2018101005. Epub 2019 Feb 20.
DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in , 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 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.
DNA 末端切除启动同源重组修复 DNA 双链断裂。MRE11-RAD50-NBS1 和磷酸化的 CtIP 执行第一步末端切除，即 MRE11 催化的核酸内切酶 DNA 切割。人类 NBS1 比其同源物 Xrs2 更为关键，突出了调节高等真核生物 MRE11 核酸酶的复杂机制。我们在重建的系统中表明，NBS1 通过其 FHA 和 BRCT 结构域作为 CtIP 磷酸化的传感器发挥作用。然后，NBS1 通过与 MRE11 的直接物理相互作用激活 MRE11-RAD50 核酸酶。在没有 NBS1 的情况下，MRE11-RAD50 表现出较弱的核酸酶活性，需要 CtIP 但不需要严格的磷酸化。这至少确定了 CtIP 增强 MRE11 的两种机制：一种是通过 NBS1 的磷酸化依赖模式，另一种是没有 NBS1 的磷酸化非依赖模式。支持这一观点的是，我们表明在缺乏 NBS1 的 FHA 和 BRCT 结构域的情况下，有限的 DNA 末端切除发生。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 期。这定义了调节 DNA 代谢中 MRE11 核酸酶的机制。
