Loss of the p12 subunit of DNA polymerase delta leads to a defect in HR and sensitization to PARP inhibitors.
Loss of the p12 subunit of DNA polymerase delta leads to a defect in HR and sensitization to PARP inhibitors.
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DOI：
10.1016/j.dnarep.2018.11.003
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发表时间：
2019-01
期刊：
影响因子：
3.8
通讯作者：
Lee MYWT
中科院分区：
文献类型：
--
作者：
Zhang S;Chao HH;Wang X;Zhang Z;Lee EYC;Lee MYWT
人类脱氧核糖核酸聚合酶δ通常以异源四聚体的形式存在于非应激、未分裂的细胞中(POLδ4)。其最小的亚基p12在紫外光的作用下以及在进入S相的过程中被瞬间降解，导致Polδ4转化为三聚体(Polδ3)。为了进一步了解这两种形式的POLδ在细胞中的具体作用，编码p12的基因(POLD4)被CRISPR/CAS9破坏，产生p12基因敲除(P12KO)细胞。因此，p12KO细胞中不存在POLδ4，使POLδ3成为POLδ活性的唯一来源。绿色荧光蛋白报告分析显示，p12KO细胞在同源重组(HR)修复中存在缺陷，表明HR需要POLδ4，而不是POLδ3。在p12KO细胞中表达标志标记的p12以恢复Polδ4可减轻HR缺陷。这些结果确立了HR修复中对POLδ4的特殊要求。这导致预测，p12KO细胞应该对化疗药物更敏感，并且应该表现出PARP抑制剂的合成致命杀伤力。P12KO细胞经顺铂和丝裂霉素C以及PARP抑制剂奥拉帕利布、他唑帕利布、鲁卡帕里布和尼拉帕利处理后的克隆性细胞存活分析证实了这些预测。Flag-p12的表达降低了H1299-p12KO细胞对PARP抑制剂的敏感性。这些发现具有临床意义，因为p12的表达水平可以作为预测肿瘤对PARP抑制剂反应的生物标志物。此外，小细胞肺癌(SCLC)表现出p12表达缺陷。对几个小细胞肺癌细胞系的分析表明，它们对PARP抑制剂表现出超敏反应，这提供了p12表达缺失可能代表HR缺乏的新的分子基础的证据。
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.
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