Loss of the p12 Subunit of DNA Polymerase Delta Leads to a Defect in HR and Sensitization to PARP Inhibitors
Biochemistry and Molecular Biology
Human DNA polymerase delta is normally present in unstressed, non-dividing cells as a heterotetramer (Pol delta4). 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 delta4 to a trimer (Pol delta3). In order to further understand the specific cellular roles of these two forms of Pol delta, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol delta4 is absent in p12KO cells, leaving Pol delta3 as the sole source of Pol delta activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol delta4, but not Pol delta3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol delta4 alleviated the HR defect. These results establish a specific requirement for Pol delta4 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.
Zhang, S., Chao, H., Wang, X., Zhang, Z., Lee, E., & Lee, M. (2019). Loss of the p12 Subunit of DNA Polymerase Delta Leads to a Defect in HR and Sensitization to PARP Inhibitors. DNA Repair, 73, 64-70. https://doi.org/10.1016/j.dnarep.2018.11.003