Molecular Toxicology Group, Department of Biology, University of Konstanz, 78457, Constance, Germany.
Cell Biol Toxicol. 2023 Feb;39(1):345-364. doi: 10.1007/s10565-022-09739-9. Epub 2022 Jul 1.
PARP1 (aka ARTD1) acts as a prime sensor of cellular genotoxic stress response. PARP1 detects DNA strand breaks and subsequently catalyzes the formation of poly(ADP-ribose) (PAR), which leads to the recruitment of the scaffold protein XRCC1 during base excision and single strand break repair and the assembly of multi-protein complexes to promote DNA repair. Here, we reveal that the recruitment of either protein to sites of DNA damage is impeded in the absence of the other, indicating a strong reciprocal relationship between the two DNA repair factors during genotoxic stress response. We further analyzed several cellular and molecular endpoints in HeLa PARP1 KO, XRCC1 KO, and PARP1/XRCC1 double KO (DKO) cells after genotoxic treatments, i.e., PARylation response, NAD levels, clonogenic survival, cell cycle progression, cell death, and DNA repair. The analysis of NAD levels and cytotoxicity after treatment with the topoisomerase I inhibitor camptothecin revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells-an effect that could be rescued by the additional genetic deletion of PARP1 as well as by pharmacological PARP inhibition. Moreover, impaired repair of hydrogen peroxide and CPT-induced DNA damage in XRCC1 KO cells could be partially rescued by additional deletion of PARP1. Our results therefore highlight important reciprocal regulatory functions of XRCC1 and PARP1 during genotoxic stress response.
PARP1（又名 ARTD1）作为细胞遗传毒性应激反应的主要传感器发挥作用。PARP1 检测 DNA 链断裂，随后催化多聚（ADP-核糖）（PAR）的形成，这导致在碱基切除和单链断裂修复过程中支架蛋白 XRCC1 的募集，以及促进 DNA 修复的多蛋白复合物的组装。在这里，我们揭示了在没有另一种蛋白的情况下，这两种蛋白都无法募集到 DNA 损伤部位，这表明在遗传毒性应激反应中，这两种 DNA 修复因子之间存在很强的相互关系。我们还在经过遗传毒性处理后，分析了 HeLa PARP1 KO、XRCC1 KO 和 PARP1/XRCC1 双 KO（DKO）细胞中的几种细胞和分子终点，即 PAR 化反应、NAD 水平、集落形成能力、细胞周期进程、细胞死亡和 DNA 修复。拓扑异构酶 I 抑制剂喜树碱处理后 NAD 水平和细胞毒性的分析显示，与 PARP1 KO 细胞相比，XRCC1 KO 细胞表现出超敏表型——这种效应可以通过 PARP1 的额外遗传缺失以及药理学 PARP 抑制来挽救。此外，XRCC1 KO 细胞中过氧化氢和 CPT 诱导的 DNA 损伤的修复受损，可以通过 PARP1 的额外缺失部分挽救。因此，我们的研究结果突出了 XRCC1 和 PARP1 在遗传毒性应激反应中的重要相互调节功能。
