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Purification and Specific Assays for Measuring APE-1 Endonuclease Activity

  • Adrian Esqueda
  • Mohammed Z. Mohammed
  • Srinivasan Madhusudan
  • Nouri NeamatiEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 928)

Abstract

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.

Key words

Apurinic and apyrimidinic endonuclease Base excision repair Drug resistance Purification Assay development Screening 

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Adrian Esqueda
    • 1
  • Mohammed Z. Mohammed
    • 1
  • Srinivasan Madhusudan
    • 1
  • Nouri Neamati
    • 2
    Email author
  1. 1.Laboratory of Molecular Oncology, School of Molecular Medical SciencesUniversity of NottinghamNottinghamUK
  2. 2.Department of Pharmacology and Pharmaceutical SciencesUniversity of Southern California, School of PharmacyLos AngelesUSA

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