Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells
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Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells. / Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav.
In: PLOS ONE, Vol. 8, No. 3, 01.01.2013, p. e59567.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells
AU - Brázdová, Marie
AU - Navrátilová, Lucie
AU - Tichý, Vlastimil
AU - Němcová, Kateřina
AU - Lexa, Matej
AU - Hrstka, Roman
AU - Pečinka, Petr
AU - Adámik, Matej
AU - Vojtesek, Borivoj
AU - Paleček, Emil
AU - Deppert, Wolfgang
AU - Fojta, Miroslav
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
KW - Binding Sites
KW - Cell Line, Tumor
KW - DNA, Superhelical
KW - Gene Expression Regulation
KW - Humans
KW - Mutant Proteins
KW - Mutation
KW - Plasmids
KW - Promoter Regions, Genetic
KW - Protein Binding
KW - Protein-Serine-Threonine Kinases
KW - Substrate Specificity
KW - Tumor Suppressor Protein p53
KW - bcl-2-Associated X Protein
U2 - 10.1371/journal.pone.0059567
DO - 10.1371/journal.pone.0059567
M3 - SCORING: Journal article
C2 - 23555710
VL - 8
SP - e59567
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 3
ER -