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.

Department of Biophysical Chemistry and Molecular Oncology Institute of Biophysics Academy of Sciences of the Czech Republic v v i Brno Czech Republic

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Petitjean A, Achatz MI, Borresen-Dale AL, Hainaut P, Olivier M (2007) TP53 mutations in human cancers: functional selection and impact on cancer prognosis and outcomes. Oncogene 26: 2157–2165. PubMed

Foord OS, Bhattacharya P, Reich Z, Rotter V (1991) A DNA binding domain is contained in the C-terminus of wild type p53 protein. Nucleic Acids Res 19: 5191–5198. PubMed PMC

Zotchev SB, Protopopova M, Selivanova G (2000) p53 C-terminal interaction with DNA ends and gaps has opposing effect on specific DNA binding by the core. Nucleic Acids Res 28: 4005–4012. PubMed PMC

Kim E, Deppert W (2003) The complex interactions of p53 with target DNA: we learn as we go. Biochem Cell Biol 81: 141–150. PubMed

Ang HC, Joerger AC, Mayer S, Fersht AR (2006) Effects of common cancer mutations on stability and DNA binding of full-length p53 compared with isolated core domains. J Biol Chem 281: 21934–21941. PubMed

Espinosa JM, Emerson BM (2001) Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol Cell 8: 57–69. PubMed

McKinney K, Mattia M, Gottifredi V, Prives C (2004) p53 linear diffusion along DNA requires its C terminus. Mol Cell 16: 413–424. PubMed

Tafvizi A, Huang F, Fersht AR, Mirny LA, van Oijen AM (2011) A single-molecule characterization of p53 search on DNA. Proc Natl Acad Sci U S A 108: 563–568. PubMed PMC

Lidor Nili E, Field Y, Lubling Y, Widom J, Oren M, et al. (2010) p53 binds preferentially to genomic regions with high DNA-encoded nucleosome occupancy. Genome Res 20: 1361–1368. PubMed PMC

Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, et al. (2002) The IARC TP53 database: new online mutation analysis and recommendations to users. Hum Mutat 19: 607–614. PubMed

Joerger AC, Fersht AR (2007) Structure-function-rescue: the diverse nature of common p53 cancer mutants. Oncogene 26: 2226–2242. PubMed

Vousden KH, Prives C (2005) P53 and prognosis: new insights and further complexity. Cell 120: 7–10. PubMed

Hinds PW, Finlay CA, Frey AB, Levine AJ (1987) Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol 7: 2863–2869. PubMed PMC

Muller P, Hrstka R, Coomber D, Lane DP, Vojtesek B (2008) Chaperone-dependent stabilization and degradation of p53 mutants. Oncogene 27: 3371–3383. PubMed

Friedlander P, Legros Y, Soussi T, Prives C (1996) Regulation of mutant p53 temperature-sensitive DNA binding. J Biol Chem 271: 25468–25478. PubMed

Gohler T, Jager S, Warnecke G, Yasuda H, Kim E, et al. (2005) Mutant p53 proteins bind DNA in a DNA structure-selective mode. Nucleic Acids Res 33: 1087–1100. PubMed PMC

Quante T, Otto B, Brazdova M, Kejnovska I, Deppert W, et al. (2012) Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity. Cell Cycle 11: 3290–3303. PubMed PMC

Walter K, Warnecke G, Bowater R, Deppert W, Kim E (2005) Tumor suppressor p53 binds with high affinity to CTG.CAG trinucleotide repeats and induces topological alterations in mismatched duplexes. J Biol Chem 280: 42497–42507. PubMed

Brazdova M, Quante T, Togel L, Walter K, Loscher C, et al. (2009) Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences. Nucleic Acids Res 37: 1486–1400. PubMed PMC

Deppert W (1996) Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions. J Cell Biochem 62: 172–180. PubMed

Deppert W, Gohler T, Koga H, Kim E (2000) Mutant p53: “gain of function” through perturbation of nuclear structure and function? J Cell Biochem Suppl Suppl 35: 115–122. PubMed

Koga H, Deppert W (2000) Identification of genomic DNA sequences bound by mutant p53 protein (Gly245–>Ser) in vivo. Oncogene 19: 4178–4183. PubMed

Blandino G, Deppert W, Hainaut P, Levine A, Lozano G, et al. (2011) Mutant p53 protein, master regulator of human malignancies: a report on the Fifth Mutant p53 Workshop. Cell Death Differ 19: 180–183. PubMed PMC

Brosh R, Rotter V (2009) When mutants gain new powers: news from the mutant p53 field. Nat Rev Cancer 9: 701–713. PubMed

Freed-Pastor WA, Prives C (2012) Mutant p53: one name, many proteins. Genes Dev 26: 1268–1286. PubMed PMC

Kim E, Deppert W (2004) Transcriptional activities of mutant p53: when mutations are more than a loss. J Cell Biochem 93: 878–886. PubMed

Kim E, Deppert W (2007) Interactions of mutant p53 with DNA: guilt by association. Oncogene 26: 2185–2190. PubMed

Zalcenstein A, Weisz L, Stambolsky P, Bar J, Rotter V, et al. (2006) Repression of the MSP/MST-1 gene contributes to the antiapoptotic gain of function of mutant p53. Oncogene 25: 359–369. PubMed

Zalcenstein A, Stambolsky P, Weisz L, Muller M, Wallach D, et al. (2003) Mutant p53 gain of function: repression of CD95(Fas/APO-1) gene expression by tumor-associated p53 mutants. Oncogene 22: 5667–5676. PubMed

Yan W, Liu G, Scoumanne A, Chen X (2008) Suppression of inhibitor of differentiation 2, a target of mutant p53, is required for gain-of-function mutations. Cancer Res 68: 6789–6796. PubMed PMC

Weisz L, Zalcenstein A, Stambolsky P, Cohen Y, Goldfinger N, et al. (2004) Transactivation of the EGR1 gene contributes to mutant p53 gain of function. Cancer Res 64: 8318–8327. PubMed

Fontemaggi G, Dell’Orso S, Trisciuoglio D, Shay T, Melucci E, et al. (2009) The execution of the transcriptional axis mutant p53, E2F1 and ID4 promotes tumor neo-angiogenesis. Nat Struct Mol Biol 16: 1086–1093. PubMed

Palecek E (1991) Local supercoil-stabilized DNA structures. Crit Rev Biochem Mol Biol 26: 151–226. PubMed

Choe CY, Kim H, Dong J, van Wijnen AJ, Law PY, et al. (2011) The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element. Gene 487: 52–61. PubMed PMC

Kouzine F, Levens D (2007) Supercoil-driven DNA structures regulate genetic transactions. Front Biosci 12: 4409–4423. PubMed

Mazur SJ, Sakaguchi K, Appella E, Wang XW, Harris CC, et al. (1999) Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain. J Mol Biol 292: 241–249. PubMed

Palecek E, Brazdova M, Brazda V, Palecek J, Billova S, et al. (2001) Binding of p53 and its core domain to supercoiled DNA. Eur J Biochem 268: 573–581. PubMed

Palecek E, Vlk D, Stankova V, Brazda V, Vojtesek B, et al. (1997) Tumor suppressor protein p53 binds preferentially to supercoiled DNA. Oncogene 15: 2201–2209. PubMed

Pivonkova H, Sebest P, Pecinka P, Ticha O, Nemcova K, et al... (2010) Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs. Biochem Biophys Res Commun. PubMed

Brazdova M, Palecek J, Cherny DI, Billova S, Fojta M, et al. (2002) Role of tumor suppressor p53 domains in selective binding to supercoiled DNA. Nucleic Acids Res 30: 4966–4974. PubMed PMC

Fojta M, Pivonkova H, Brazdova M, Nemcova K, Palecek J, et al. (2004) Investigations of the supercoil-selective DNA binding of wild type p53 suggest a novel mechanism for controlling p53 function. Eur J Biochem 271: 3865–3876. PubMed

Jagelska EB, Pivonkova H, Fojta M, Brazda V (2010) The potential of the cruciform structure formation as an important factor influencing p53 sequence-specific binding to natural DNA targets. Biochem Biophys Res Commun 391: 1409–1414. PubMed

Palecek E, Brazda V, Jagelska E, Pecinka P, Karlovska L, et al. (2004) Enhancement of p53 sequence-specific binding by DNA supercoiling. Oncogene 23: 2119–2127. PubMed

Midgley CA, Fisher CJ, Bartek J, Vojtesek B, Lane D, et al. (1992) Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli. J Cell Sci 101 (Pt 1): 183–189. PubMed

Klein C, Georges G, Kunkele KP, Huber R, Engh RA, et al. (2001) High thermostability and lack of cooperative DNA binding distinguish the p63 core domain from the homologous tumor suppressor p53. J Biol Chem 276: 37390–37401. PubMed

Sif S, Gilmore TD (1994) Interaction of the v-Rel oncoprotein with cellular transcription factor Sp1. J Virol 68: 7131–7138. PubMed PMC

Shi XB, Nesslinger NJ, Deitch AD, Gumerlock PH, deVere White RW (2002) Complex functions of mutant p53 alleles from human prostate cancer. Prostate 51: 59–72. PubMed

Bowater R, Aboul-Ela F, Lilley DM (1992) Two-dimensional gel electrophoresis of circular DNA topoisomers. Methods Enzymol 212: 105–120. PubMed

Brazdova M, Kizek R, Havran L, Palecek E (2002) Determination of glutathione-S-transferase traces in preparations of p53 C-terminal domain (aa320–393). Bioelectrochemistry 55: 115–118. PubMed

Pivonkova H, Brazdova M, Kasparkova J, Brabec V, Fojta M (2006) Recognition of cisplatin-damaged DNA by p53 protein: critical role of the p53 C-terminal domain. Biochem Biophys Res Commun 339: 477–484. PubMed

Palecek E, Brazdova M, Cernocka H, Vlk D, Brazda V, et al. (1999) Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments. Oncogene 18: 3617–3625. PubMed

Thornborrow EC, Manfredi JJ (1999) One mechanism for cell type-specific regulation of the bax promoter by the tumor suppressor p53 is dictated by the p53 response element. J Biol Chem 274: 33747–33756. PubMed

Rohaly G, Chemnitz J, Dehde S, Nunez AM, Heukeshoven J, et al. (2005) A novel human p53 isoform is an essential element of the ATR-intra-S phase checkpoint. Cell 122: 21–32. PubMed

Powell DJ, Hrstka R, Candeias M, Bourougaa K, Vojtesek B, et al. (2008) Stress-dependent changes in the properties of p53 complexes by the alternative translation product p53/47. Cell Cycle 7: 950–959. PubMed

Nemcova K, Havran L, Sebest P, Brazdova M, Pivonkova H, et al. (2010) A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads. Anal Chim Acta 668: 166–170. PubMed

Vikhanskaya F, Lee MK, Mazzoletti M, Broggini M, Sabapathy K (2007) Cancer-derived p53 mutants suppress p53-target gene expression–potential mechanism for gain of function of mutant p53. Nucleic Acids Res 35: 2093–2104. PubMed PMC

Hebbar PB, Archer TK (2008) Altered histone H1 stoichiometry and an absence of nucleosome positioning on transfected DNA. J Biol Chem 283: 4595–4601. PubMed PMC

Rustighi A, Tessari MA, Vascotto F, Sgarra R, Giancotti V, et al. (2002) A polypyrimidine/polypurine tract within the Hmga2 minimal promoter: a common feature of many growth-related genes. Biochemistry 41: 1229–1240. PubMed

Tomonaga T, Levens D (1996) Activating transcription from single stranded DNA. Proc Natl Acad Sci U S A 93: 5830–5835. PubMed PMC

Kim E, Rohaly G, Heinrichs S, Gimnopoulos D, Meissner H, et al. (1999) Influence of promoter DNA topology on sequence-specific DNA binding and transactivation by tumor suppressor p53. Oncogene 18: 7310–7318. PubMed

Oren M, Rotter V (2010) Mutant p53 Gain-of-Function in Cancer. Cold Spring Harb Perspect Biol 2: a001107. PubMed PMC

Kaku S, Albor A, Kulesz-Martin M (2001) Dissociation of DNA binding and in vitro transcriptional activities dependent on the C terminus of P53 proteins. Biochem Biophys Res Commun 280: 204–211. PubMed

Grochova D, Vankova J, Damborsky J, Ravcukova B, Smarda J, et al. (2008) Analysis of transactivation capability and conformation of p53 temperature-dependent mutants and their reactivation by amifostine in yeast. Oncogene 27: 1243–1252. PubMed

MAR-WIZ website. Available: http://genomecluster.secs.oakland.edu/marwiz/. Accessed 2013 Feb 28.

Bi C, Benham CJ (2004) WebSIDD: server for predicting stress-induced duplex destabilized (SIDD) sites in superhelical DNA. Bioinformatics 20: 1477–1479. PubMed

Cer RZ, Bruce KH, Mudunuri US, Yi M, Volfovsky N, et al. (2011) Non-B DB: a database of predicted non-B DNA-forming motifs in mammalian genomes. Nucleic Acids Res 39: D383–391. PubMed PMC

Lexa M, Martinek T, Burgetova I, Kopecek D, Brazdova M (2011) A dynamic programming algorithm for identification of triplex-forming sequences. Bioinformatics 27: 2510–2517. PubMed

Cherny DI, Brazdova M, Palecek J, Palecek E, Jovin TM (2005) Sequestering of p53 into DNA-protein filaments revealed by electron microscopy. Biophys Chem 114: 261–271. PubMed

O’Farrell TJ, Ghosh P, Dobashi N, Sasaki CY, Longo DL (2004) Comparison of the effect of mutant and wild-type p53 on global gene expression. Cancer Res 64: 8199–8207. PubMed

Scian MJ, Stagliano KE, Deb D, Ellis MA, Carchman EH, et al. (2004) Tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes. Oncogene 23: 4430–4443. PubMed

Solomon H, Buganim Y, Kogan-Sakin I, Pomeraniec L, Assia Y, et al. (2012) Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature. J Cell Sci 125: 3144–3152. PubMed

van Oijen MG, Slootweg PJ (2000) Gain-of-function mutations in the tumor suppressor gene p53. Clin Cancer Res 6: 2138–2145. PubMed

Weisz L, Oren M, Rotter V (2007) Transcription regulation by mutant p53. Oncogene 26: 2202–2211. PubMed

Schilling T, Kairat A, Melino G, Krammer PH, Stremmel W, et al. (2010) Interference with the p53 family network contributes to the gain of oncogenic function of mutant p53 in hepatocellular carcinoma. Biochem Biophys Res Commun 394: 817–823. PubMed

Sinha S, Malonia SK, Mittal SP, Singh K, Kadreppa S, et al. (2010) Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element. Embo J 29: 830–842. PubMed PMC

Vikhanskaya F, Siddique MM, Kei Lee M, Broggini M, Sabapathy K (2005) Evaluation of the combined effect of p53 codon 72 polymorphism and hotspot mutations in response to anticancer drugs. Clin Cancer Res 11: 4348–4356. PubMed

Stros M, Ozaki T, Bacikova A, Kageyama H, Nakagawara A (2002) HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73-dependent sequence-specific transactivation from the human Bax gene promoter. J Biol Chem 277: 7157–7164. PubMed

Thornborrow EC, Manfredi JJ (2001) The tumor suppressor protein p53 requires a cofactor to activate transcriptionally the human BAX promoter. J Biol Chem 276: 15598–15608. PubMed

Yoshihara Y, Wu D, Kubo N, Sang M, Nakagawara A, et al. (2012) Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response. Biochem Biophys Res Commun 421: 57–63. PubMed

Will K, Warnecke G, Wiesmuller L, Deppert W (1998) Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing. Proc Natl Acad Sci U S A 95: 13681–13686. PubMed PMC

Strano S, Dell’Orso S, Di Agostino S, Fontemaggi G, Sacchi A, et al. (2007) Mutant p53: an oncogenic transcription factor. Oncogene 26: 2212–2219. PubMed

Muller PA, Caswell PT, Doyle B, Iwanicki MP, Tan EH, et al. (2009) Mutant p53 drives invasion by promoting integrin recycling. Cell 139: 1327–1341. PubMed

Xu J, Reumers J, Couceiro JR, De Smet F, Gallardo R, et al. (2011) Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol 7: 285–295. PubMed

Albor A, Kaku S, Kulesz-Martin M (1998) Wild-type and mutant forms of p53 activate human topoisomerase I: a possible mechanism for gain of function in mutants. Cancer Res 58: 2091–2094. PubMed

Gobert C, Skladanowski A, Larsen AK (1999) The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53. Proc Natl Acad Sci U S A 96: 10355–10360. PubMed PMC

Soe K, Grosse F (2003) p53 stimulates human topoisomerase I activity by modulating its DNA binding. Nucleic Acids Res 31: 6585–6592. PubMed PMC

El-Hizawi S, Lagowski JP, Kulesz-Martin M, Albor A (2002) Induction of gene amplification as a gain-of-function phenotype of mutant p53 proteins. Cancer Res 62: 3264–3270. PubMed

Gatz SA, Wiesmuller L (2006) p53 in recombination and repair. Cell Death Differ 13: 1003–1016. PubMed

Markham NR, Zuker M (2008) UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453: 3–31. PubMed

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