聚合酶 epsilon (Polϵ) 在单链断裂 (SSB) 上进行的前导链 DNA 复制会导致单端双链断裂 (seDSB)，单端双链断裂 (seDSB) 可通过同源定向修复 (HDR) 进行修复，并通过叉反转 (FR) 进行抑制。尽管之前的研究确定了羟基脲诱导的 FR 所需的许多分子，但对 seDSB 的 FR 知之甚少。在这里，我们鉴定了在 seDSB 上特异性介导 FR 的分子。由于 seDSB 的 FR 需要聚（ADP 核糖）聚合酶 1 (PARP1)，因此我们假设 seDSB/FR 相关分子会增加对喜树碱 (CPT) 的耐受性，但不会增加对 PARP 抑制剂奥拉帕尼的耐受性，尽管两种抗癌药物都会产生 seDSB。事实上，我们发现 Polϵ 核酸外切酶和 CTF18（一种 Polϵ 辅因子）可以增加对 CPT 的耐受性，但不能增加奥拉帕尼的耐受性。为了探索 Polϵ 核酸外切酶、CTF18 和 PARP1 之间潜在的功能相互作用，我们创建了核酸外切酶缺陷的 POLE1exo-/-、CTF18-/-、PARP1-/-、CTF18-/-/POLE1exo-/-、PARP1-/-/POLE1exo -/- 和 CTF18-/-/PARP1-/- 细胞。上位性分析表明 Polϵ 核酸外切酶和 CTF18 是相互依赖的，并且需要 PARP1 来实现 CPT 耐受。值得注意的是，POLE1exo-/- 和 HDR 缺陷的 BRCA1-/- 细胞表现出相似的 CPT 敏感性。此外，将 POLE1exo-/- 与 BRCA1-/- 突变相结合可协同提高 CPT 敏感性。总之，新鉴定的 PARP1-CTF18-Polϵ 核酸外切酶轴和 HDR 独立发挥作用，防止 seDSB 处的叉塌陷。奥拉帕尼抑制该轴，解释了奥拉帕尼对 HDR 缺陷细胞的显着细胞毒性作用。© 作者 2023。由牛津大学出版社代表核酸研究出版。

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.