Mapping the Ku Interactome Using Proximity-Dependent Biotin Identification in Human Cells

The Ku heterodimer, composed of Ku70 and Ku80, is best characterized for its role in repairing double-stranded DNA breaks but is also known to participate in other regulatory processes. Despite our understanding of Ku protein interplay during DNA repair, the extent of Ku’s protein interactions in other processes has never been fully determined. Using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS) with wild-type Ku70, we identified candidate proteins that interact with the Ku heterodimer in HEK293 cells, in the absence of exogenously induced DNA damage. BioID analysis identified approximately 250 nuclear proteins, appearing in at least two replicates, including known Ku-interacting factors such as MRE11A, WRN, and NCOA6. Meanwhile, AP-MS analysis identified approximately 50 candidate proteins. Of the novel protein interactors identified, many were involved in functions already suspected to involve Ku such as transcriptional regulation, DNA replication, and DNA repair, while several others suggest that Ku may be involved in additional functions such as RNA metabolism, chromatin-remodeling, and microtubule dynamics. Using a combination of BioID and AP-MS, this is the first report that comprehensively characterizes the Ku protein interaction landscape, revealing new cellular processes and protein complexes involving the Ku complex.Ku异二聚体（Ku heterodimer）由Ku70与Ku80构成，其最广为人知的功能是修复双链DNA断裂（double-stranded DNA breaks），同时也被证实参与多种其他调控过程。尽管学界对Ku蛋白在DNA修复过程中的相互作用机制已有一定认知，但Ku蛋白在其他生理过程中的蛋白质相互作用范围仍未被完全阐明。本研究借助依赖于邻近连接的生物素标记鉴定（proximity-dependent biotin identification, BioID）以及亲和纯化结合质谱（affinity purification coupled to mass spectrometry, AP-MS）技术，以野生型Ku70为靶标，在未施加外源诱导DNA损伤的HEK293细胞（HEK293 cells）中鉴定出与Ku异二聚体相互作用的候选蛋白。BioID分析共鉴定出约250种核蛋白，且至少在两次生物学重复中均被检测到，其中包含已知的Ku相互作用因子如MRE11A、WRN与NCOA6。与此同时，AP-MS分析共鉴定出约50种候选蛋白。在本次鉴定得到的新型互作蛋白中，多数功能与此前推测的Ku参与的生物学过程相契合，例如转录调控、DNA复制及DNA修复；另有部分因子提示Ku可能还参与RNA代谢、染色质重塑以及微管动力学等全新的生物学过程。本研究结合BioID与AP-MS技术，首次全面刻画了Ku复合物的蛋白质相互作用全景图谱，揭示了涉及Ku复合物的全新细胞进程与蛋白质复合物。