Exonuclease 1 (Exo1) Participates in Mammalian Non-Homologous End Joining and Contributes to Drug Resistance in Ovarian Cancer.

ABSTRACT

BACKGROUND Exonuclease 1 (Exo1) participates in a variety of DNA damage repair, including mismatch repair, nucleotide excision repair, and homologous recombination. Genetic study in yeast indicates a role of Exo1 in non-homologous end joining (NHEJ), acting as a regulator for accuracy repairing DNA. This study aimed to investigate the effects of human Exo1 in NHEJ and drug resistance in ovarian cells. MATERIAL AND METHODS Ectopic expression of Exo1 was carried out using pcDNA3.1-EXO1 plasmid in SKOV3 cells. GST-tagged human Exo1 was purified using pTXB1-gst-EXO1 and the his-tagged-Ku was collected using pET15b.his.Ku. Exo1 and Ku70 proteins expressed in bacteria were harvested and purified. DNA-protein binding was examined using affinity capture assay. The cells were treated using drugs for 72 hours. Then, the viabilities of cells were evaluated with sulforhodamine B cell viability analysis. The protein expression was evaluated using western blot assay. RESULTS As expected, human cells that deficient of Exo1 were sensitive to ionizing radiation and DNA damaging drugs (cisplatin and doxorubicin). Cisplatin resistant ovarian cancer cell line and Exo1 deficient cell lines were successfully generated. Exo1 interacts with NHEJ required factor Ku70 and affects NHEJ efficiency. We observed that Exo1 expression level was upregulated in drug resistant cell line and knockdown of Exo1 in drug resistant cells sensitized cells to cisplatin and doxorubicin. CONCLUSIONS Exo1 participated in mammalian non-homologous end joining and contributed to drug resistance in ovarian cancer.