Rahman Md Ratul, Kawasumi Ryotaro, Hirota Kouji
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1,Hachioji-shi, Tokyo 192-0397, Japan.
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1,Hachioji-shi, Tokyo 192-0397, Japan.
DNA Repair (Amst). 2024 Dec;144:103773. doi: 10.1016/j.dnarep.2024.103773. Epub 2024 Oct 9.
Remdesivir is a 1'-cyano-modified adenine nucleotide analog used for the treatment of COVID-19. Recently, the anti-carcinogenic effect of remdesivir has been also identified in human cancers. However, the impact of this drug and the mechanisms underlying the cellular tolerance to remdesivir have not been elucidated. Here, we explored DNA repair pathways responsible for the cellular tolerance to remdesivir by monitoring the sensitivity of 24 mutant DT40 cells deficient in various DNA repair pathways. We found that cells deficient in FEN1 displayed the highest sensitivity against remdesivir. Since FEN1 contributes to base excision repair (BER), we measured the cellular sensitivity to remdesivir in mutants deficient in BER and found that other BER mutants such as XRCC1 and PARP1 cells are tolerant to remdesivir, indicating that FEN1 contributes to cellular tolerance to remdesivir through roles other than BER. We observed augmented DNA damage and acute cell cycle arrest at early S-phase after remdesivir treatment in FEN1 cells. Moreover, the replication fork progression was significantly slowed by remdesivir in FEN1 cells, indicating a direct involvement of FEN1 in replication fork progression when replication is challenged by remdesivir. Since FEN1 contributes to Okazaki fragment maturation (OFM), a process ligating Okazaki fragments generated during lagging strand synthesis, we analyzed the kinetics of the repair of single-strand breaks (SSBs) in nascent DNA. Strikingly, FEN1 cells exhibited slowed kinetics in OFM, and remdesivir incorporation critically impaired this process in FEN1 cells. These results indicate that remdesivir is preferentially incorporated in Okazaki fragments leading to the failure of Okazaki fragment maturation and FEN1 plays a critical role in suppressing remdesivir-mediated DNA damage through Okazaki fragment processing. Collectively, we revealed a previously unappreciated role of FEN1 in the cellular tolerance to remdesivir.
瑞德西韦是一种用于治疗新型冠状病毒肺炎（COVID-19）的1'-氰基修饰的腺嘌呤核苷酸类似物。最近，瑞德西韦的抗癌作用也在人类癌症中得到了确认。然而，这种药物的影响以及细胞对瑞德西韦产生耐受性的潜在机制尚未阐明。在此，我们通过监测24种缺乏各种DNA修复途径的突变型DT40细胞的敏感性，探索了负责细胞对瑞德西韦耐受性的DNA修复途径。我们发现，缺乏FEN1的细胞对瑞德西韦表现出最高的敏感性。由于FEN1参与碱基切除修复（BER），我们测量了缺乏BER的突变体对瑞德西韦的细胞敏感性，发现其他BER突变体如XRCC1和PARP1细胞对瑞德西韦具有耐受性，这表明FEN1通过BER以外的作用机制对细胞对瑞德西韦的耐受性起作用。我们观察到，在FEN1细胞中，瑞德西韦处理后DNA损伤增加，并且在S期早期出现急性细胞周期停滞。此外，瑞德西韦显著减缓了FEN1细胞中复制叉的进展，这表明当复制受到瑞德西韦挑战时，FEN1直接参与复制叉的进展。由于FEN1参与冈崎片段成熟（OFM），即连接滞后链合成过程中产生的冈崎片段的过程，我们分析了新生DNA中单链断裂（SSB）修复的动力学。令人惊讶的是，FEN1细胞在OFM过程中表现出较慢的动力学，并且瑞德西韦的掺入严重损害了FEN1细胞中的这一过程。这些结果表明，瑞德西韦优先掺入冈崎片段，导致冈崎片段成熟失败，并且FEN1在通过冈崎片段加工抑制瑞德西韦介导的DNA损伤中起关键作用。总的来说，我们揭示了FEN1在细胞对瑞德西韦耐受性方面以前未被认识到的作用。
