PARP1通过Poly(ADP-ribose)化作用（PARylation）或PARP1和Poly(ADP-ribose)葡萄聚糖酶（PARG）通过dePARylation起着DNA损伤响应的动态调控同等重要的作用。作为最活跃的dePARylation酶，PARG通过pADPr依赖和PCNA依赖的机制被招募到DNA损伤位点。靶向dePARylation被认为是克服PARP抑制剂抗药性的一种替代策略。然而，dePARylation在正常不受干扰的细胞中的功能仍然不清楚。为解决这一挑战，我们进行了多次CRISPR筛选，并揭示了S期pADPr的PARG介导的dePARylation对细胞存活至关重要。失去dePARylation活性最初引起了S期特异的pADPr信号，这是由于未连接的冈萨基片段，最终导致了pADPr的无控制积累和依赖于PARP1/2的细胞毒性。此外，我们证明参与冈萨基片段连接和/或碱基切除修复的蛋白质调控pADPr信号和PARG抑制引起的细胞死亡。此外，我们确定PARG的表达对细胞对PARG抑制的敏感性至关重要。此外，我们揭示了PARG通过抑制pADPr来维持细胞存活至关重要。总之，我们的数据不仅确定了PARG在正常增殖细胞中的重要作用，还为进一步发展PARG抑制剂的癌症治疗提供了潜在生物标志物。 Poly(ADP-ribosyl)ation是一种可逆的翻译后修饰。虽然PARG可能对过度的PARP1参与具有保护作用，但是关于PARG作用机制的详细知识仍然不清楚。在这里，我们展示了PARG参与DNA复制，尤其是在冈萨基片段成熟中的作用。此外，PARG水平对细胞对PARG抑制的敏感性至关重要，这是PARGi基于疗法的一个宝贵生物标志物。

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.Poly(ADP-ribosyl)ation is a reversible post-translational modification. Although PARG may have a protective effect against excessive PARP1 engagement, detailed knowledge of PARG's mechanism of action remains elusive. Here, we showed that PARG participates in DNA replication, especially in Okazaki fragment maturation. Moreover, PARG level is critically important for cellular sensitivity to PARG inhibition, which is a valuable biomarker for PARGi-based therapy.