Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Faculty of Natural Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland.
Mol Cell. 2017 Oct 19;68(2):431-445.e5. doi: 10.1016/j.molcel.2017.09.019. Epub 2017 Oct 12.
Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage.
Mec1-Ddc2（ATR-ATRIP）是一种关键的 DNA 损伤感应激酶，通过单链（ss）DNA 结合复制蛋白 A（RPA）募集，以启动 DNA 损伤检查点反应。在没有 DNA 损伤的情况下激活 ATR-ATRIP 是致命的。因此，损伤特异性募集先于激酶激活是很重要的，这至少部分是通过 Mec1-Ddc2 同源二聚体实现的。在这里，我们报告了酵母 Mec1-Ddc2-RPA 组装的结构、生化和功能特征。Ddc2-Rfa1 和 Ddc2-Rfa1-t11（K45E 突变体）N 末端的高分辨率共晶结构以及 Ddc2 卷曲螺旋结构域（CCD）的结构提供了对 Mec1-Ddc2 同源二聚体和损伤部位靶向的深入了解。基于我们的结构和功能发现，我们提出了一个 Mec1-Ddc2-RPA-ssDNA 复合结构模型。通过验证，我们表明 RPA 依赖性募集 Mec1-Ddc2 对于维持其在 ssDNA 上的同源二聚体状态至关重要，并且 Ddc2 的募集结构域和 CCD 对于 Mec1 依赖性生存至关重要紫外线诱导的 DNA 损伤。
