Abstract
Reactive oxygen species (ROS) overproduction results in oxidative stress leading to genomic instability via the generation of small base lesions in the genome, and this unrepaired DNA base damage leads to various cellular consequences. The oxidative stress-mediated DNA base damage is involved in various human disorders like cancer, cardiovascular, ocular, and neurodegenerative diseases. Base excision repair (BER) pathway, one of the DNA repair pathways, is majorly involved in the repair of oxidative DNA base lesions, which utilizes a different set of enzymes, including endonuclease viz Apurinic/apyrimidinic endonuclease 1 (APE1). APE1 is a well-known multifunctional enzyme with DNA repair, REDOX regulatory, and protein-protein interaction/cross-talk functions associated with the cell survival mechanisms. APE1 acts as an important player in both normal and cancerous cell survival; thus, evaluating its endonuclease activity in the biological samples provide useful readout of the DNA repair capacity/ability, which can be used to tune for the development of therapeutic candidates via either stimulating or blocking its DNA repair function in normal vs. cancer cells, respectively. This chapter enlists two methods used for the determination of APE1’s endonuclease activity by oligonucleotide-based radioactive P32-labeled and nonradioactive fluorescence dyes using the cell extracts and recombinant APE1 protein.
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Acknowledgments
This work was supported to A.K.M. by the funds recieved under BSR-start-up grant from the University Grants Commission (UGC), New Delhi, India, and the funds received under the scheme Research Seed Money (RSM) from the Central University of Punjab, Bathinda (CUPB). K.B.G and S.K. acknowledge financial support in the form of a senior research fellowship (SRF) from the Indian Council for Medical Research (ICMR) and University Grants Commission (UGC), Govt. of India, New Delhi, India. The Central Instrumentation Laboratory (CIL), CUPB, is thankfully acknowledged. Dr. Bibyendu Banerjee, Senior Scientist, Central Drug Research Institute (CDRI), Lucknow, India, is thankfully acknowledged for the technical training rendered for the fluorescence-based assays to K.B.G. Because of the limited focus of this article, many relevant and appropriate references could not be included, for which the authors apologize.
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Gupta, K.B., Kaur, S., Dhiman, M., Mantha, A.K. (2022). Methods to Assess Oxidative DNA Base Damage Repair of Apurinic/Apyrimidinic (AP) Sites Using Radioactive and Nonradioactive Oligonucleotide-Based Assays. In: Deep, G. (eds) Cancer Biomarkers. Methods in Molecular Biology, vol 2413. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1896-7_16
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DOI: https://doi.org/10.1007/978-1-0716-1896-7_16
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