Nbs1 将人 Mre11/Rad50 核酸酶复合物转化为特异性针对蛋白-DNA 加合物的内切/外切核酸酶机器。

Nbs1 Converts the Human Mre11/Rad50 Nuclease Complex into an Endo/Exonuclease Machine Specific for Protein-DNA Adducts.

机构信息

Howard Hughes Medical Institute and Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.

出版信息

Mol Cell. 2016 Nov 3;64(3):593-606. doi: 10.1016/j.molcel.2016.10.010.

DOI:10.1016/j.molcel.2016.10.010

PMID:27814491

Abstract

The human Mre11/Rad50/Nbs1 (hMRN) complex is critical for the sensing, processing, and signaling of DNA double-strand breaks. The nuclease activity of Mre11 is essential for mammalian development and cell viability, although the regulation and substrate specificity of Mre11 have been difficult to define. Here we show that hMRN catalyzes sequential endonucleolytic and exonucleolytic activities on both 5' and 3' strands of DNA ends containing protein adducts, and that Nbs1, ATP, and adducts are essential for this function. In contrast, Nbs1 inhibits Mre11/Rad50-catalyzed 3'-to-5' exonucleolytic degradation of clean DNA ends. The hMRN endonucleolytic cleavage events are further stimulated by the phosphorylated form of the human C-terminal binding protein-interacting protein (CtIP) DNA repair enzyme, establishing a role for CtIP in regulating hMRN activity. These results illuminate the important role of Nbs1 and CtIP in determining the substrates and consequences of human Mre11/Rad50 nuclease activities on protein-DNA lesions.

摘要

人源 Mre11/Rad50/Nbs1（hMRN）复合物对于 DNA 双链断裂的感应、处理和信号转导至关重要。尽管 Mre11 的酶活性对于哺乳动物的发育和细胞活力至关重要，但它的调控和底物特异性一直难以确定。在这里，我们展示 hMRN 可以在含有蛋白加合物的 DNA 末端的 5'和 3'链上催化连续的内切核酸酶和外切核酸酶活性，并且 Nbs1、ATP 和加合物对于该功能是必需的。相比之下，Nbs1 抑制 Mre11/Rad50 催化的无蛋白 DNA 末端的 3'-5'外切核酸酶降解。hMRN 的内切核酸酶切割事件进一步受到人 C 端结合蛋白相互作用蛋白（CtIP）DNA 修复酶磷酸化形式的刺激，这确立了 CtIP 在调节 hMRN 活性中的作用。这些结果阐明了 Nbs1 和 CtIP 在决定人源 Mre11/Rad50 核酸酶活性在蛋白-DNA 损伤上的底物和后果方面的重要作用。