SpringerProtocols: Abstract: The Use of Electrophoretic Mobility Shift Assays to Study DNA Repair

The Use of Electrophoretic Mobility Shift Assays to Study DNA Repair

By: Byung Joon Hwang², Vaughn Smider², Gilbert Chu²

Abstract

Full Text

Download PDF (161K)

DNA repair pathways must include proteins that recognize and bind to damaged DNA. The search for such proteins has been facilitated by the use of electrophoretic mobility shift assays (EMSAs), which were first used to detect transcription factors that bind to specific DNA sequences (1,2). To study DNA repair, EMSAs have been adapted to detect proteins that bind to specific DNA lesions or structures.

Affiliation(s): (2) Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, CA

Book Title: DNA Repair Protocols: Eukaryotic Systems

Series: Methods in Molecular Biology | Volume: 113 | Pub. Date: Jun-21-1999 | Page Range: 103-120 | DOI: 10.1385/1-59259-675-4:103

Subject: Cell Biology

References

Download References

1.	Fried, M. and Crothers, D. M. (1981) Equilibrium and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525.

2.	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.

3.	Chu, G. and Chang, E. (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science 242, 564–567.

4.	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.

5.	Hwang, B. J. and Chu, G. (1993) Purification and characterization of a protein that binds to damaged DNA. Biochemistry 32, 1657–1666.

6.	Hwang, B. J., Liao, J., and Chu, G. (1996) Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutat. Res. 362, 105–117.

7.	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.

8.	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.

9.	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.

10.	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.

11.	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.

12.	Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestly, A., Demengeot, J., Mizuta, R., et al. (1994) Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265, 1442–1445.

13.	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.

14.	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.

15.	Jiricny, J. (1994) Colon cancer and DNA repair: have mismatches met their match? Trends Genet. 10, 164–168. <Occurrence Type="DOI"><Handle>10.1016/0168-9525(94)90093-0</Handle></Occurrence>

16.	Donahue, B. A., Augot, M., Bellon, S. F., Treiber, D. K., Toney, J. H., Lippard, S. J., 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.

17.	Toney, J., Donahue, B., Kellett, P., Bruhn, S., Essigmann, J., and Lippard, S. (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.

18.	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.

19.	Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

20.	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. <Occurrence Type="DOI"><Handle>10.1016/0003-2697(76)90527-3</Handle></Occurrence>

21.	Payne, A and Chu, G. (1994) Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage. Mutat. Res. 310, 89–102.

Comments (Loading...)

Post comment/View all comments