Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage
Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage
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DOI：
10.1016/j.molcel.2017.09.019
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发表时间：
2017-10-19
期刊：
影响因子：
16
通讯作者：
Gasser, Susan M.
中科院分区：
文献类型：
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作者：
Deshpande, Ishan;Seeber, Andrew;Gasser, Susan M.
Mec 1-Ddc 2（ATR-ATRIP）是一种重要的DNA损伤敏感激酶，通过单链DNA结合复制蛋白A（RPA）募集，启动DNA损伤检查点反应。在没有DNA损伤的情况下，ATR-ATRIP的激活是致命的。因此，重要的是损伤特异性募集先于激酶活化，这至少部分地通过Mec 1-Ddc 2同源二聚化来实现。在这里，我们报告的结构，生化和功能特性的酵母Mec 1-Ddc 2-RPA大会。Ddc 2-Rfa 1和Ddc 2-Rfa 1-t11（K45 E突变体）N末端和Ddc 2卷曲螺旋结构域（CCD）的高分辨率共晶体结构提供了对Mec 1-Ddc 2同源二聚化和损伤位点靶向的深入了解。基于我们的结构和功能的研究结果，我们提出了一个Mec 1-Ddc 2-RPA-ssDNA复合结构模型。通过验证，我们表明，RPA依赖的招聘Mec 1-Ddc 2是至关重要的保持其同源二聚体状态的ssDNA和Ddc 2的招聘结构域和CCD是重要的Mec 1依赖的生存UV光诱导的DNA损伤。
Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damagesensing kinase that is recruited through the singlestranded (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. Highresolution 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.