Abstract
The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to the components of the E. coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3059.
Fried, M. and Crothers, D. M. (1981) Equilibrium and kinetics of lac repressoroperator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525.
Tang, J. and Chu, G. (2002) Xeroderma pigmentosum complementation group E and UV-damaged DNA-binding protein. DNA Repair 1, 601–616.
Smider, V. and Chu, G. (1997) The end-joining reaction in V(D)J recombination. Semin. Immunol. 9, 189–197.
Chu, G. and Chang, E. (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science 242, 564–567.
Singh, H., Sen, R., Baltimore, D., and Sharp, P. (1986) A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature 319, 154–158.
Hwang, B. J. and Chu, G. (1993) Purification and characterization of a protein that binds to damaged DNA. Biochemistry 32, 1657–1666.
Hwang, B. J., Liao, J., and Chu, G. (1996) Isolation of a cDNA encoding a UVdamaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutat. Res. 362, 105–117.
Chu, G. and Chang, E. (1990) Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA. Proc. Natl. Acad. Sci. USA 87, 3324–3327.
Keeney, S., Eker, A. P. M., Brody, T., Vermeulen, W., Bootsma, D. and Hoeijmakers, J. H. J. (1994) Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein. Proc. Natl. Acad. Sci. USA 91, 4053–4056.
Tang, J., Hwang, B. J., Ford, J. M., Hanawalt, P. C., and Chu, G. (2000) Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis. Mol. Cell 5, 737–744.
Rathmell, W. K. and Chu, G. (1994) A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol. Cell. Biol. 14, 4741–4748.
Rathmell, W. K. and Chu, G. (1994) Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc. Natl. Acad. Sci. USA 91, 7623–7627.
Smider, V., Rathmell, W. K., Lieber, M., and Chu, G. (1994) Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 266, 288–291.
Taccioli, G. E., Gottlieb, T. M., Blunt, T., et al. (1994) Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265, 1442–1445.
Hammarsten, O. and Chu, G. (1998) DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. Proc. Natl. Acad. Sci. USA 95, 525–530.
Patterson, M. and Chu, G. (1989) Evidence that xeroderma pigmentosum cells from complementation group E are deficient in a homolog of yeast photolyase. Mol. Cell. Biol. 9, 5105–5112.
Fox, M., Feldman, B., and Chu, G. (1994) A novel role for DNA photolyase: binding to drug-induced DNA damage is associated with enhanced cytotoxicity in yeast. Mol. Cell. Biol. 14, 8071–8077.
Jiricny, J. (1994) Colon cancer and DNA repair: have mismatches met their match? Trends Genetics 10, 164–168.
Donahue, B.A., Augot, M., Bellon, S. F., et al. (1990) Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin. Biochemistry 29, 5872–5880.
Toney, J., Donahue, B. A., Kellett, P. J., Bruhn, S. L., Essigmann, J. M., Lippard, S. J. (1989) Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II). Proc. Natl. Acad. Sci. USA 86, 8328–8332.
Brown, S., Kellet, P., and Lippard, S. (1993) Ixr1, a yeast protein that binds to platinated DNA and confers sensitivity to cisplatin. Science 261, 603–605.
Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Payne, A. and Chu, G. (1994) Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage. Mutat. Res. 310, 89–102.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Smider, V., Hwang, B.J., Chu, G. (2006). Electrophoretic Mobility Shift Assays to Study Protein Binding to Damaged DNA. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:323
Download citation
DOI: https://doi.org/10.1385/1-59259-973-7:323
Publisher Name: Humana Press
Print ISBN: 978-1-58829-513-2
Online ISBN: 978-1-59259-973-8
eBook Packages: Springer Protocols