title: Electrophoretic mobility shift assays for protein-DNA complexes involved in DNA repair.
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 (35)S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein-DNA complex.
authors.affiliation: Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, CA, USA.
authors.forename: Chun
authors.initials: C
authors.lastname: Tsai
authors.forename: Vaughn
authors.initials: V
authors.lastname: Smider
authors.forename: Byung Joon
authors.initials: BJ
authors.lastname: Hwang
authors.forename: Gilbert
authors.initials: G
authors.lastname: Chu
mesh_terms: D002021:Buffers; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D002945:Cisplatin / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage; D015342:DNA Probes / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D002021:Buffers; D015342:DNA Probes; D004268:DNA-Binding Proteins; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005090:Exodeoxyribonucleases; D002945:Cisplatin