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。