Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells

PLoS One. 2013;8(3):e59567. doi: 10.1371/journal.pone.0059567. Epub 2013 Mar 26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line, Tumor
  • DNA, Superhelical / chemistry
  • DNA, Superhelical / metabolism*
  • Gene Expression Regulation / genetics
  • Humans
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation*
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein-Serine-Threonine Kinases / genetics
  • Substrate Specificity
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • bcl-2-Associated X Protein / genetics


  • DNA, Superhelical
  • Mutant Proteins
  • Tumor Suppressor Protein p53
  • bcl-2-Associated X Protein
  • STK4 protein, human
  • Protein-Serine-Threonine Kinases

Grant support

This work was supported by the Czech Science Foundation (P301/10/2370, 13-36108S, 204/06/P369 and 204/08/1560, to MB; P301/11/1678 to BV; P301/11/2055 to EP), by the Internal Grant Agency of the Ministry of Health of the CR (NT/13794-4/2012 and RECAMO CZ. 1.05/2.1.00/03.0101 to BV), by the Ministry of Education of the CR (1K04119 to MB), by the Academy of Sciences of the CR (AV0Z50040702) and by the European commission (QLGA-CT-2001-52001 and MERG-6-CT-2005-014875 to MB, EU - 502983 to EP and to WD) and is a part of the project: Institute of Environmental Technologies, reg. no. CZ.1.05/2.1.00/03.0100. The senior professorship of WD is financially supported by the Jung Foundation for Science, Hamburg. The publication reflects only the authors’ views and the Community is not liable for any use that may be made of the information contained therein. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.