体内外多种信号会导致DNA损伤，DNA损伤应答和修复是维持真核细胞基因组稳定性和完整性的关键。其失调导致多种疾病，尤其是肿瘤发生。DNA损伤应答关键蛋白ATM、ATR、DNA-PKcs同属于PIKK蛋白激酶家族，三个激酶分子量大并分别形成多种复合物，在DNA损伤应答过程中起关键作用。申请人前期解析了DNA-PK全酶复合物结构（Cell Res，2017），ATR-ATRIP复合物结构（Cell Res，2018），初步回答了相关复合物组装和激酶活性调节机制。申请人拟在此基础上，深入研究ATM、ATR、DNA-PKcs及相关复合物的结构与功能，解析相关复合物的冷冻电镜结构，结合生化分析，系统的揭示激酶活性调节、复合物组装、及这些蛋白参与DNA损伤应答动态过程的分子机制。项目的顺利完成，将为DNA损伤应答的基础研究和靶向DNA损伤应答的药物开发提供重要的结构基础。

DNA damage can be induced by intrinsic or extrinsic stimuli and may lead to genomic instability, tumorigenesis and other cellular abnormalities. Although DNA double-strand breaks (DSBs) do not occur as frequently as the other lesions, they are difficult to repair and extremely toxic. .ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit) are three master regulators of the DNA-damage response (DDR), which coordinates a number of important cellular processes and is essential for the maintenance of genome integrity and stability in living organisms. The DDR downstream pathways include cell cycle arrest, apoptosis, DNA damage repair, gene transcription, etc. The DDR pathways have been extensively studied during the past two decades. However, the three-dimensional structures of the key protein complexes remain largely unknown due to technical challenges..ATM and DNA-PKcs primarily respond to DSBs, whereas ATR is activated by DNA damages like DSBs, crosslinks, and DNA replication stress. ATM, ATR, and DNA-PKcs are giant proteins kinases, which belong to the family of phosphoinositide-3-kinase-related kinases (PIKKs) and share high sequence homology and functional similarity. We have previously reported cryo-EM structures of DNA-PK holoenzyme (DNA-PKcs, KU80, KU70, and DNA) (Cell Res, 2017) and ATR-ATRIP complex (Cell Res, 2018). The studies provide structural insight into the mechanisms for complex assembly, regulation of the kinase activity of DNA-PKcs and ATR. .Here, we propose to further investigate the structures and functions of ATM, ATR, and DNA-PKcs, and related protein complexes. We want to use cyro-EM to determine the structures of the related proteins and protein complexes, and perfrom biochemical analyses to further investigate the underlying mechanism. We believe that our studies would provide structural basis for understanding the dynamic processes of DNA damage response.