Tumor Suppressor Genes pp 87-100

Part of the Methods in Molecular Biology™ book series (MIMB, volume 223)

Electrophoretic Mobility Shift Analysis of the DNA Binding of Tumor Suppressor Gene Products

  • Edward C. Thornborrow
  • Matthew Maurer
  • James J. Manfredi


Central to the tumor suppressor activity of certain proteins is the ability to interact physically with DNA. A well-studied example of this is the tumor suppressor p53 (1,2). The p53 protein has been implicated in several diverse growth-related pathways, including apoptosis and cell cycle arrest (3,4) and the p53 gene is mutated in the majority of human cancers (5,6). At its amino-terminus, the protein contains a potent transcriptional activation domain (7) which is linked to a central core domain that mediates sequence-specific DNA binding (8, 9, 10). Both of these domains have been shown to be important for p53-mediated growth suppression (11). The relevance of the DNA-binding domain to the tumor suppressor activity of p53 is further highlighted by the fact that the major sites of mutation found in human cancers are localized to this region (12). Several of these mutations have been shown to abolish the ability of p53 to function as a transcriptional activator (13, 14, 15). A DNA consensus sequence through which p53 binds and activates transcription has been identified. This sequence consists of two palindromic decamers of 5′-RRRCWWGYYY-3′ (where R is a purine, Y is a pyrimidine, and W is an adenine or thymine) separated by 0–13 bp (16, 17, 18). Through sequences similar to this consensus, p53 has been shown to activate the transcription of many genes, including bax, p21, mdm2, gadd45, IGF-BP3, and cyclin G (19, 20, 21, 22, 23, 24, 25, 26), which leads to the various physiologic outcomes that contribute to the ability of p53 to function as a tumor suppressor.


  1. 1.
    Bargonetti, J., Friedman, P. N., Kern, S. E., Vogelstein, B., and Prives, C. (1991) Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell 65, 1083–1091.PubMedCrossRefGoogle Scholar
  2. 2.
    Kern, S. E., Kinzler, K. W., Bruskin, A., et al. (1991) Identification of p53 as a sequence-specific DNA-binding protein. Science 252, 1708–1711.PubMedCrossRefGoogle Scholar
  3. 3.
    Ko, L. J. and Prives, C. (1996) p53: puzzle and paradigm. Genes Dev. 10, 1054–1072.PubMedCrossRefGoogle Scholar
  4. 4.
    Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.PubMedCrossRefGoogle Scholar
  5. 5.
    Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878.PubMedGoogle Scholar
  6. 6.
    Hollstein, M., Rice, K., Greenblatt, M. S., et al. (1994) Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22, 3551–3555.PubMedGoogle Scholar
  7. 7.
    Fields, S. and Jang, S. K. (1990) Presence of a potent transcription activating sequence in the p53 protein. Science 249, 1046–1049.PubMedCrossRefGoogle Scholar
  8. 8.
    Bargonetti, J., Manfredi, J. J., Chen, X., Marshak, D. R., and Prives, C. (1993) A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein. Genes Dev. 7, 2565–2574.PubMedCrossRefGoogle Scholar
  9. 9.
    Pavletich, N. P., Chambers, K. A., and Pabo, C. O. (1993) The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots. Genes Dev. 7, 2556–2564.PubMedCrossRefGoogle Scholar
  10. 10.
    Wang, Y., Reed, M., Wang, P., et al. (1993) p53 domains: identification and characterization of two autonomous DNA-binding regions. Genes Dev. 7, 2575–2586.PubMedCrossRefGoogle Scholar
  11. 11.
    Pietenpol, J. A., Tokino, T., Thiagalingam, S., el-Deiry, W. S., Kinzler, K. W., and Vogelstein, B. (1994) Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc. Natl. Acad Sci. USA 91, 1998–2002.PubMedCrossRefGoogle Scholar
  12. 12.
    Prives, C. (1994) How loops, beta sheets, and alpha helices help us to understand p53. Cell 78, 543–546.PubMedCrossRefGoogle Scholar
  13. 13.
    Kern, S. E., Pietenpol, J. A., Thiagalingam, S., Seymour, A., Kinzler, K. W., and Vogelstein, B. (1992) Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 256, 827–830.PubMedCrossRefGoogle Scholar
  14. 14.
    Raycroft, L., Wu, H. Y., and Lozano, G. (1990) Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 249, 1049–1051.PubMedCrossRefGoogle Scholar
  15. 15.
    Unger, T., Nau, M. M., Segal, S., and Minna, J. D. (1992) p53: a transdominant regulator of transcription whose function is ablated by mutations occurring in human cancer. EMBO J. 11, 1383–1390.PubMedGoogle Scholar
  16. 16.
    El-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., and Vogelstein, B. (1992) Definition of a consensus binding site for p53. Nat. Genet. 1, 45–49.PubMedCrossRefGoogle Scholar
  17. 17.
    Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., and Shay, J. W. (1992) A transcriptionally active DNA-binding site for human p53 protein complexes. Mol. Cell. Biol. 12, 2866–2871.PubMedGoogle Scholar
  18. 18.
    Halazonetis, T. D., Davis, L. J., and Kandil, A. N. (1993) Wild-type p53 adopts a “mutant”-like conformation when bound to DNA. EMBO J. 12, 1021–1028.PubMedGoogle Scholar
  19. 19.
    Buckbinder, L., Talbott, R., Velasco-Miguel, S., et al. (1995) Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377, 646–649.PubMedCrossRefGoogle Scholar
  20. 20.
    El-Deiry, W. S., Tokino, T., Velculescu, V. E., et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825.PubMedCrossRefGoogle Scholar
  21. 21.
    El-Deiry, W. S., Tokino, T., Waldman, T., et al. (1995) Topological control of p21WAF1/CIP1 expression in normal and neoplastic tissues. Cancer Res. 55, 2910–2919.PubMedGoogle Scholar
  22. 22.
    Hollander, M. C., Alamo, I., Jackman, J., Wang, M. G., McBride, O. W., and Fornace, A. J., Jr. (1993) Analysis of the mammalian gadd45 gene and its response to DNA damage. J. Biol. Chem. 268, 24385–24393.PubMedGoogle Scholar
  23. 23.
    Macleod, K. F., Sherry, N., Hannon, G., et al. (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 9, 935–944.PubMedCrossRefGoogle Scholar
  24. 24.
    Miyashita, T. and Reed, J. C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299.PubMedCrossRefGoogle Scholar
  25. 25.
    Perry, M. E., Piette, J., Zawadzki, J. A., Harvey, D., and Levine, A. J. (1993) The mdm-2 gene is induced in response to UV light in a p53-dependent manner. Proc. Natl. Acad. Sci. USA 90, 11623–11627.PubMedCrossRefGoogle Scholar
  26. 26.
    Zauberman, A., Lupo, A., and Oren, M. (1995) Identification of p53 target genes through immune selection of genomic DNA: the cyclin G gene contains two distinct p53 binding sites. Oncogene 10, 2361–2366.PubMedGoogle Scholar
  27. 27.
    Ceglarek, J. A. and Revzin, A. (1989) Studies of DNA-protein interactions by gel electrophoresis. Electrophoresis 10, 360–365.PubMedCrossRefGoogle Scholar
  28. 28.
    Revzin, A. (1989) Gel electrophoresis assays for DNA-protein interactions. Biotechniques 7, 346–355.PubMedGoogle Scholar
  29. 29.
    Thornborrow, E. C. and Manfredi, J. J. (2001) The tumor suppressor protein p53 requires a cofactor to transcriptionally activate the human bax promoter. J. Biol. Chem. 276, 15,598–15,608.PubMedCrossRefGoogle Scholar
  30. 30.
    Resnick-Silverman, L., St Clair, S., Maurer, M., Zhao, K., and Manfredi, J. J. (1998) Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53. Genes Dev. 12, 2102–2107.PubMedCrossRefGoogle Scholar
  31. 31.
    Thornborrow, E. C., and Manfredi, J. J. (1999) One mechanism for cell type-specific regulation of the bax promoter by the tumor suppressor p53 is dictated by the p53 response element. J. Biol. Chem. 274, 33747–33756.PubMedCrossRefGoogle Scholar
  32. 32.
    Lassar, A. B., Davis, R. L., Wright, W. E., et al. (1991) Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell 66, 305–315.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Edward C. Thornborrow
    • 1
  • Matthew Maurer
    • 1
  • James J. Manfredi
    • 1
  1. 1.Derald H. Ruttenberg Cancer CenterMount Sinai School of MedicineNew York

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