• MeSH
• Biosensing Techniques instrumentation   methods   MeSH
• Electrochemical Techniques instrumentation   methods   MeSH
• Glycomics instrumentation   methods   MeSH
• Glycoproteins analysis   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Proteins analysis   metabolism   MeSH
• Carbohydrate Sequence MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Glycoproteins MeSH
• Proteins MeSH

A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON) using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear) exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS) was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations≥200 mM under the same conditions. These observations suggest: (a) different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b) a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

• MeSH
• Potassium Chloride pharmacology   MeSH
• Nucleic Acid Conformation MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Plasmids chemistry   metabolism   MeSH
• Salts pharmacology   MeSH
• Protein Binding drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium Chloride MeSH
• Tumor Suppressor Protein p53 MeSH
• Salts MeSH

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.

• MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Humans MeSH
• Mutation * MeSH
• Mutant Proteins chemistry   genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 chemistry   genetics   metabolism   MeSH
• Plasmids genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• bcl-2-Associated X Protein genetics   MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• Substrate Specificity MeSH
• DNA, Superhelical chemistry   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Intracellular Signaling Peptides and Proteins MeSH
• Mutant Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• bcl-2-Associated X Protein MeSH
• Protein Serine-Threonine Kinases MeSH
• STK4 protein, human MeSH Browser
• DNA, Superhelical MeSH