Department of Biochemistry, Western University, London, ON, Canada.
Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.
Sci Rep. 2023 Jul 27;13(1):12162. doi: 10.1038/s41598-023-39166-7.
The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.
Ku70/80 异二聚体是非同源末端连接 DNA 修复的关键因子，但它也参与其他细胞功能，如端粒调控和维持，而 Ku 在这些功能中的作用尚未完全确定。此前有报道称，敲除人细胞系中的 Ku80 会导致细胞死亡，但 Ku 在人细胞中必需的根本原因尚未得到充分探索。在这里，我们使用 CRISPR/Cas9 编辑建立了条件性 Ku70 敲除细胞，以研究 Ku70 功能的必需性。虽然我们观察到 Ku 耗竭时细胞活力丧失，但我们没有检测到端粒长度的显著变化，也没有在 Ku 缺失时记录到致命水平的 DNA 损伤。Ku70 耗竭后对全蛋白质组变化的分析显示，包括细胞周期/有丝分裂、RNA 相关过程和翻译/核糖体生物发生在内的几个细胞途径失调。我们的研究表明，Ku70 敲除细胞活力丧失的驱动原因与 Ku 在 DNA 修复或端粒中的功能无关。此外，我们的数据表明 Ku 缺失会影响多个细胞过程和途径，并表明 Ku 在维持细胞活力方面在 DNA 修复和端粒维持以外的细胞过程中发挥着关键作用。
