Purification and Specific Assays for Measuring APE-1 Endonuclease Activity

Human apurinic/apyrimidinic endonuclease-1 (APE-1) is essential for base excision repair and plays a major role in DNA repair and maintaining genomic stability. Cancer cells treated with conventional DNA-damaging agents develop resistance due in part to upregulation of enzymes involved in DNA repair. It is hypothesized that inhibiting DNA repair machinery should sensitize the cells to DNA-damaging agents. Previously, it has been shown that APE-1 is implicated in drug resistance and cancer progression. Therefore, APE-1 inhibitors are being sought after for their synergistic properties with various chemotherapeutics agents. Screening of several compound libraries and optimization of known inhibitors of APE-1 endonuclease activity have been accelerated by the use of high-throughput screening. Nevertheless, potential inhibitors must be tested in other counterscreens to validate their selectivity for APE-1. Here, we describe in-depth protocols for APE-1 purification and development of assays specific for APE-1 endonuclease activity.