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Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage
Molecular Cell ( IF 14.5 ) Pub Date : 2017-10-12 , DOI: 10.1016/j.molcel.2017.09.019
Ishan Deshpande , Andrew Seeber , Kenji Shimada , Jeremy J. Keusch , Heinz Gut , Susan M. Gasser

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.



中文翻译:

Mec1-Ddc2-RPA组装的结构基础以及在损伤位点对单链DNA的激活

Mec1-Ddc2(ATR-ATRIP)是一种关键的DNA损伤敏感激酶,可通过单链(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,重要的是损伤特异性募集先于激酶激活,这至少部分是通过Mec1-Ddc2均二聚作用实现的。在这里,我们报告酵母Mec1-Ddc2-RPA大会的结构,生化和功能表征。Ddc2-Rfa1和Ddc2-Rfa1-t11(K45E突变体)N末端和Ddc2卷曲螺旋结构域(CCD)的高分辨率共晶体结构提供了对Mec1-Ddc2同型二聚化和损伤部位靶向的洞察力。根据我们的结构和功能发现,我们提出了Mec1-Ddc2-RPA-ssDNA复合结构模型。

更新日期:2017-10-12
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