title: Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells.
abstract: 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.
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic. maruska@ibp.cz
authors.forename: Marie
authors.initials: M
authors.lastname: Brázdová
authors.forename: Lucie
authors.initials: L
authors.lastname: Navrátilová
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Tichý
authors.forename: Kateřina
authors.initials: K
authors.lastname: Němcová
authors.forename: Matej
authors.initials: M
authors.lastname: Lexa
authors.forename: Roman
authors.initials: R
authors.lastname: Hrstka
authors.forename: Petr
authors.initials: P
authors.lastname: Pečinka
authors.forename: Matej
authors.initials: M
authors.lastname: Adámik
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojtesek
authors.forename: Emil
authors.initials: E
authors.lastname: Paleček
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Deppert
authors.forename: Miroslav
authors.initials: M
authors.lastname: Fojta
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics; D000099317:Hippo Kinases
chemical_list: D004278:DNA, Superhelical; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D017346:Protein Serine-Threonine Kinases; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C095566:STK4 protein, human; D000099317:Hippo Kinases
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