The p53 protein is an unusually shaped tetramer that binds directly to DNA.

Abstract

We have analyzed the size and structure of native immunopurified human p53 protein. By using a combination of chemical crosslinking, gel ifitration chromatography, and zonal velocity gradient centrifugation, we have determined that the predominant form of p53 in such preparations is a tetramer. The behavior of purified p53 in gels and sucrose gradients implies that the protein has an extended shape. Wild-type p53 has been shown to bind specifically to sites in cellular and viral DNA. We show in this study by Southwestern ligand blotting and by analysis of DNA-bound crosslinked p53 that p53 monomers, dimers, and tetramers can bind directly to DNA.As alterations in the function of the p53 gene product may be involved in a wide variety of human cancers, it is imperative to gain insight into this important protein. p53 contains four highly conserved regions that are 90-100% homologous among the diverse vertebrate species examined (1). Strikingly, the great majority of missense mutations identified in diverse human tumor types are located in the conserved blocks within p53 (reviewed in ref. 2). These data suggest that these highly conserved regions of p53 are crucial for the correct functioning of the wild-type protein. In addition to the importance of the conserved regions of p53, key features of the p53 polypeptide reside in its more peripheral regions as well. The NH2 portion of the p53 protein was shown to include a strong transcriptional activation domain (3-6). The COOH portion contains both primary and secondary nuclear localization signals (7,8), as well as sequences that contribute to p53 protein-protein interactions (9, 10) and protein-DNA interactions (11).Important clues to the function of p53 are derived from studies showing that p53 binds site-specifically to ) was applied to a TSK 3000 gel filtration column previously equilibrated with column buffer (100 mM sodium phosphate, pH 7.07/1 mM EDTA) and run on the Pharmacia FPLC system. Elution was carried out at 30 ml/hr, and eluted samples were collected in 1-ml fractions and absorbance readings were confirmed by both SDS/PAGE and Bio-Rad dye-binding protein assay. Protein molecular weight standards (1 mg/ml) were run and analyzed in parallel.Sucrose Gradient Centrifugation. Immunoaffinity-purified p53 (2 ,g) in 500 ,ul of 50 mM Tris-HCl, pH 8.0/150 mM NaCl/1% (wt/vol) Nonidet P-40/1 mM dithiothreitol was layered directly on a gradient of 5-20% (wt/vol) sucrose (5 ml) in phosphate-buffered saline (PBS: 8 mM Na2HPO4/1.5 mM KH2PO4/140 mM NaCl/3 mM KCl, pH 7.5). Gradients were centrifuged in a Beckman SW50.1 rotor at 25,000 rpm for 16 hr at 4°C. Fractions (-300 IlI) were collected and subjected to SDS/PAGE after precipitation with 5% (wt/vol) trichloroacetic acid in the presence of 20 ,ug of bovine serum albumin as carrier. Proteins were electrotransferred to nitrocellulose and immunostained with a combination of p53-specific antibodies Pab 421 and Pab 1801.DNA Binding. Southwestern analysis of p53 proteins was as described (11). Duplicate samples of...

Camptothecin-induced downregulation of MLL5 contributes to the activation of tumor suppressor p53

“…Interestingly, in the p53 mutants with mutations potentially affecting its DNA binding capability, including R175H (conformational mutant), R248W (DNA-binding mutant), and S392A, all three mutants were still able to interact with MLL5 (Supplementary Figure S12). In the nucleus, equilibrium exists between monomers, dimers and tetramers of p53 (Friedman et al, 1993;Chene, 2001). We speculate that in the presence of chromatin-bound MLL5, p53 tetramers are excluded from the chromatin and the equilibrium is shifted towards dimers or monomers that can shuttle to cytoplasm for degradation ( Figure 8a).…”

p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor

“…p53 molecules can oligomerize to form a homotetramer (Friedman et al, 1993), and this association is likely to be required for e cient transcription activity. However, the ability to oligomerize can also lead to inactivation of the wild type protein complexed with a mutant protein (Deb et al, 1999).…”

p53 regulation by post-translational modification and nuclear retention in response to diverse stresses

“…This appears to be due to the binding of the peptides to the p53 NES such that when these p53/peptide heterocomplexes enter the nucleus, they may be incapable of nuclear export because the binding surface recognized by the export receptor is hidden by the peptide. Tetrameric p53 is most e ective at binding and transactivating p53 response elements, and mutations which prevent tetramerization compromise DNA binding, diminish transactivation, and lead to tumorigenesis (Friedman et al, 1993;Hainaut et al, 1994;Halazonetis and Kandil, 1993;Hupp and Lane, 1994;Ishioka et al, 1995;Lomax et al, 1998;McLure and Lee, 1998;Pietenpol et al, 1994;Tarunina et al, 1996;Varley et al, 1996). Therefore, stress-induced modi®cations which allow p53 tetramerization to occur may concurrently contribute to both the formation of DNA-binding tetramers and to their retention in the nucleus.…”