Protocol

Tumor Suppressor Genes

Volume 223 of the series Methods in Molecular Biology™ pp 87-100

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

  • Edward C. ThornborrowAffiliated withDerald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine
  • , Matthew MaurerAffiliated withDerald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine
  • , James J. ManfrediAffiliated withDerald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine

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Abstract

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 (810). 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 (1315). 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 (1618). 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 (1926), which leads to the various physiologic outcomes that contribute to the ability of p53 to function as a tumor suppressor.