GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity

作者：Yang Han, Hongling Zhao, Gang Li, Jia Jin, Hejiang Guo, Jinpeng Tan, Xingyao Sun, Saiyu Li, Qian Ran, Chenjun Bai, Yongqing Gu, Zhongjun Li, Hua Guan, Shanshan Gao, Ping‐Kun Zhou · 发表于：British Journal of Cancer · 年份：2024 · DOI：10.1038/s41416-024-02636-4 · 被引用次数：22 · 研究领域：DNA Repair Mechanisms、PARP inhibition in cancer therapy、Histone Deacetylase Inhibitors Research

BACKGROUND: DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. METHODS: Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. RESULTS: Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembl...