DNA Repair Protocols pp 53-78

Part of the Methods in Molecular Biology book series (MIMB, volume 920)

Electrophoretic Mobility Shift Assays for Protein–DNA Complexes Involved in DNA Repair

  • Chun Tsai
  • Vaughn Smider
  • Byung Joon Hwang
  • Gilbert Chu
Protocol

Abstract

The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA–PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein–DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein–DNA complex.

Key words

UV-DDB Global genomic repair Nucleotide excision repair Ku DNA-PKcs XRCC4/Ligase IV DNA-PK Nonhomologous end joining Double-strand break repair V(D)J recombination 

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Chun Tsai
    • 1
  • Vaughn Smider
    • 2
  • Byung Joon Hwang
    • 3
  • Gilbert Chu
    • 1
  1. 1.Departments of Medicine and BiochemistryStanford University Medical CenterStanfordUSA
  2. 2.Department of Molecular BiologyThe Scripps Research InstituteLa JollaUSA
  3. 3.Department of Molecular Bioscience, College of Biomedical ScienceKangwon National UniversityChuncheonRepublic of Korea

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