Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, United States.
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, United States.
DNA Repair (Amst). 2019 Jan;73:64-70. doi: 10.1016/j.dnarep.2018.11.003. Epub 2018 Nov 13.
Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ4 to a trimer (Pol δ3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ4 is absent in p12KO cells, leaving Pol δ3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ4 alleviated the HR defect. These results establish a specific requirement for Pol δ4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency.
人类 DNA 聚合酶 δ 在未受应激的非分裂细胞中通常以异四聚体（Pol δ4）的形式存在。其最小亚基 p12 会在 UV 损伤以及进入 S 期时短暂降解，导致 Pol δ4 转化为三聚体（Pol δ3）。为了进一步了解这两种形式的 Pol δ 的特定细胞作用，通过 CRISPR/Cas9 破坏编码 p12 的基因（POLD4），以产生 p12 敲除（p12KO）细胞。因此，p12KO 细胞中不存在 Pol δ4，使得 Pol δ3 成为唯一的 Pol δ 活性来源。GFP 报告基因实验表明，p12KO 细胞同源重组（HR）修复存在缺陷，表明 Pol δ4（而非 Pol δ3）是 HR 所必需的。在 p12KO 细胞中表达 Flag 标记的 p12 以恢复 Pol δ4 可缓解 HR 缺陷。这些结果确立了 Pol δ4 在 HR 修复中的特定需求。这导致预测 p12KO 细胞对化疗药物更敏感，并且应该通过 PARP 抑制剂表现出合成致死性杀伤。p12KO 细胞用顺铂和丝裂霉素 C 以及 PARP 抑制剂奥拉帕利、他拉唑帕利、鲁卡帕利和尼拉帕利处理的克隆形成细胞存活实验证实了这些预测。H1299-p12KO 细胞对 PARP 抑制剂的敏感性通过表达 Flag-p12 得到缓解。这些发现具有临床意义，因为 p12 的表达水平可能是肿瘤对 PARP 抑制剂反应的预测生物标志物。此外，小细胞肺癌（SCLC）已知存在 p12 表达缺陷。对几种 SCLC 细胞系的分析表明，它们对 PARP 抑制剂高度敏感，这为 p12 表达缺失可能代表 HR 缺陷的新分子基础提供了证据。
