Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex
Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex
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DOI：
10.1074/jbc.m102946200
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发表时间：
2001-07-13
影响因子：
4.8
通讯作者：
Karnitz, LM
中科院分区：
文献类型：
--
作者：
Burtelow, MA;Roos-Mattjus, PMK;Karnitz, LM
DNA损伤激活细胞周期检查点信号通路，协调细胞周期阻滞和DNA修复。免疫沉淀和酵母双杂交研究表明，参与检查点信号传导的三种蛋白Rad1、Hus1和Rad9相互作用。然而，目前尚不清楚这些蛋白质如何相互作用并组装成复合物。在目前的研究中，我们证明了在人类细胞中，所有的hRad9和hHus1以及大约一半的hRad1细胞池作为一个稳定的、生物化学上离散的复合物相互作用，表观分子质量为160 kDa。这三种重组蛋白在异源体系中共表达，重组复合物与内源复合物表现出相同的色谱行为。相互作用研究使用差异标记的蛋白质表明，蛋白质不自聚合。相反，每个蛋白都有一个与其他两个伙伴的结合位点，hRad9的N端与hRad1相互作用，hRad1的N端与hHus1相互作用，hHus1的N端与hRad9预测的pcna样区域的C端相互作用。总的来说，这些分析提出了这三种蛋白质如何组装形成功能性检查点复合物的模型，我们将其称为9-1-1复合物。
DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.