Abstract
AP endonuclease 1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. APE1 possesses DNA repair activity as an endonuclease that hydrolyzes the phosphodiester bond just 5' to an apurinic/apyrimidinic (AP) site. In addition to its role in DNA repair, APE1 regulates the expression of genes important for cellular viability, and cancer promotion and progression by stimulating the DNA binding activity of various transcription factors. Previous studies in which APE1 was knocked down using siRNA revealed that tumor cells were induced apoptosis and increased the sensitivity to chemotherapy agents. However, these knockdown studies did not fully elucidate the significance of APE1 in the BER pathway. In this study, we prepared APE1 knockdown cells which are responsive to Doxycycline using Tet-On® System (Clontech) to examine the effect of APE1 knocking down on the BER pathway. We initially prepared pSingle-tTs-shRNA (6,990 bp, Clontech) inserted the target sequence of human APE1 (NCBI:NM001641). Then we transfected the plasmid into HeLa RC355 cells and selected the transfectant on the basis of the resistance to G418. We are going to examine the sensivity to methylmethane sulfonate (MMS) and H2O2.
