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• W1973291880 abstract "The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the “activation” phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of “latent” and “activated” p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions. The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the “activation” phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of “latent” and “activated” p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions. The tumor suppressor p53 is a DNA-binding protein with several DNA binding activities. Of those, sequence-specific DNA binding is the most important one because it mediates the transcriptional activity of p53 (1Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2129) Google Scholar, 2Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5744) Google Scholar). Sequence specificity of DNA binding is determined by the recognition of sequences that share homology to the consensus sequence 5′-(RRRC(A/T)(T/A)GYYY)n-3′, where R is a purine nucleotide, and Y is a pyrimidine nucleotide (3El-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1736) Google Scholar). Although sequence-specific DNA binding of p53 has been analyzed in great detail since its discovery in 1991 (4Kern S.E. Kinzler K.W. Bruskin A. Jarosz D. Friedman P. Prives C. Vogelstein B. Science. 1991; 252: 1708-1711Crossref PubMed Scopus (934) Google Scholar), the detailed molecular interaction of p53 with its target sequences in promoter elements is still a matter of debate. In particular, the question of whether sequence-specific DNA binding of p53 requires an activation step is discussed controversially (Ref. 5Ahn J. Prives C. Nat. Struct. Biol. 2001; 8: 730-732Crossref PubMed Scopus (100) Google Scholar; for review, see Ref. 6Kim E. Deppert W. Biochem. Cell Biol. 2003; 81: 141-150Crossref PubMed Scopus (55) Google Scholar). Earlier observations had indicated that unmodified p53 seems to be inactive for sequence-specific DNA binding, whereas various posttranslational modifications in the p53 C-terminal domain, binding of the monoclonal antibody PAb421 that recognizes an epitope within the p53 C terminus, or deletion of the 30 C-terminal amino acids strongly enhance sequence-specific DNA binding under certain in vitro conditions (7Hupp T.R. Lane D.P. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 195-206Crossref PubMed Scopus (106) Google Scholar). The data imply that the C terminus negatively regulates sequence-specific DNA binding of p53 (8Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (859) Google Scholar). The inhibiting effects of the C terminus were explained by the “conformation” model, which postulates that the p53 protein exists in two distinct conformations termed “latent” (for DNA binding inactive p53) and “activated” (for DNA binding active p53). According to this model, the C-terminal domain in “latent” p53 directly interacts with the core domain, thereby inhibiting sequence-specific DNA binding of p53 (8Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (859) Google Scholar). A conformational switch that converts latent p53 into an “activated” form relieves the allosteric inhibition. However, recent evidence strongly argues against the conformation model, because the overall conformation of p53 in a DNA binding active or inactive form seems to be quite similar (9Ayed A. Mulder F.A.A. Yi G.-S. Lu Y. Kay L.E. Arrowsmith C.H. Nat. Struct. Biol. 2001; 8: 756-760Crossref PubMed Scopus (230) Google Scholar). Although the latency concept could also be explained by other models (like e.g. the “competition” or “interference” model (10Anderson M.E. Woelker B. Reed M. Wang P. Tegtmeyer P. Mol. Cell. Biol. 1997; 17: 6255-6264Crossref PubMed Scopus (104) Google Scholar), the most critical argument against p53 latency is that activation of p53 does not seem to be required in vivo (11Kaeser M.D. Iggo R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 95-100Crossref PubMed Scopus (271) Google Scholar) or in different in vitro settings (11Kaeser M.D. Iggo R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 95-100Crossref PubMed Scopus (271) Google Scholar, 12Kim E. Albrechtsen N. Deppert W. Oncogene. 1997; 15: 857-869Crossref PubMed Scopus (56) Google Scholar, 13Kim E. Rohaly G. Heinrichs S. Gimnopoulos D. Meibner H. Deppert W. Oncogene. 1999; 18: 7310-7318Crossref PubMed Scopus (45) Google Scholar). Activation thus seems to be restricted to certain in vitro assays.Several features have been delineated that might account for the apparent discrepancies, like type of binding assay (12Kim E. Albrechtsen N. Deppert W. Oncogene. 1997; 15: 857-869Crossref PubMed Scopus (56) Google Scholar, 14Espinosa J. Emerson B. Mol. Cell. 2001; 8: 57-69Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar) and structure of the target DNA (12Kim E. Albrechtsen N. Deppert W. Oncogene. 1997; 15: 857-869Crossref PubMed Scopus (56) Google Scholar, 15Cain C. Miller S. Ahn J. Prives C. J. Biol. Chem. 2000; 275: 39944-39953Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 16Göhler T. Reimann M. Cherny D. Walter K. Warnecke G. Kim E. Deppert W. J. Biol. Chem. 2002; 277: 41192-41203Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In particular, the need for activation of p53 for sequence-specific DNA binding seems to be restricted to the interaction of p53 with short linear DNA and the application of electrophoretic mobility shift assay (EMSA) 1The abbreviations used are: EMSA, electrophoretic mobility shift assay; FCS, fluorescence correlation spectroscopy; TAMRA, 6-carboxytetramethylrhodamine; CRD, C-terminal regulatory domain; DBD, DNA binding domain.1The abbreviations used are: EMSA, electrophoretic mobility shift assay; FCS, fluorescence correlation spectroscopy; TAMRA, 6-carboxytetramethylrhodamine; CRD, C-terminal regulatory domain; DBD, DNA binding domain. for the analysis of sequence-specific DNA binding. Therefore, the concern has been raised that the “activation phenomenon” may be an artifact (11Kaeser M.D. Iggo R.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 95-100Crossref PubMed Scopus (271) Google Scholar, 14Espinosa J. Emerson B. Mol. Cell. 2001; 8: 57-69Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). However, numerous laboratories have reproducibly observed the activation phenomenon in various assays. Therefore, its disqualification as an “artifact” could be shortsighted, as it might reflect an important and biologically relevant feature of p53-DNA interactions that, for some reasons, is only revealed under certain experimental conditions.To further address the issue of p53 latency and activation, we employed EMSA and fluorescence correlation spectroscopy (FCS) to the analysis of the binding of p53 to double-stranded DNA oligonucleotides. FCS is based on the measurement of translational diffusion of fluorescent-labeled molecules through a confocal detection volume (10–15 liter). The method focuses on the detection of single particles rather than on averages over large numbers of particles as conventional macroscopic fluorescence detection methods. The fluorescence emission from the small detection volume is recorded in a time-resolved manner. Thus, the fluorescence quanta that belong to one fluorescing species can easily be identified by auto-correlating the time-resolved signals. Autocorrelation is a function of the diffusion times and the fractions of the different fluorescing species (17Eigen M. Rigler R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5740-5747Crossref PubMed Scopus (902) Google Scholar, 18Weiss S. Science. 1999; 283: 1676-1683Crossref PubMed Scopus (1814) Google Scholar). For the setup used in FCS readers, the optimal concentration range of the fluorescing species is between 1 and 10 nm, guaranteeing a good fluctuation of the fluorescence signal. At higher concentrations the spontaneous fluorescence fluctuation decreases and, thus, the measured signal as well. Differences in size and/or shape of free (unbound) or protein-bound fluorescent-labeled DNA molecules result in different translational diffusion times, thereby allowing quantitative analysis of protein-DNA interactions (e.g. K D and K I measurements) in solution. Specifically, the DNA-bound p53 protein (∼192 kDa for the p53 tetramer) can be quantitatively discriminated from unbound oligonucleotide DNA (∼20 kDa) in solution because of a 10-fold difference in molecular mass. Importantly, FCS enables a quantitative analysis of p53-DNA interactions in the absence or presence of various modifiers of p53 DNA binding without the need to “quench” unspecific DNA interactions using competitor DNA.Here we provide evidence that latent and activated p53 are able to bind to short double-stranded DNA oligonucleotides with high affinity. However, high affinity DNA binding of latent p53 is not sequence-specific. Sequence-specific recognition of the target DNA requires activation of p53 by PAb421. Activation is accompanied by a moderate increase in binding affinity for specific DNA and a significant drop in the affinity for nonspecific DNA. We propose that high affinity sequence-specific and nonspecific interactions of p53 with DNA require different modes of DNA recognition by the p53 core domain that are regulated by the p53 C terminus.EXPERIMENTAL PROCEDURESProtein Purification—Recombinant p53 proteins (human and mouse) expressed in insect cells were isolated as described in Bessard et al. (19Bessard A.C.H. Garay E. Lacronique V. Legros Y. Demarquay C. Houque A. Portefaix J.M. Granier C. Soussi T. Oncogene. 1998; 16: 883-890Crossref PubMed Scopus (18) Google Scholar) and purified by ion-exchange chromatography as described previously (20Janus F. Albrechtsen N. Knippschild U. Wiesmueller L. Grosse F. Deppert W. Mol. Cell. Biol. 1999; 19: 2155-2168Crossref PubMed Scopus (44) Google Scholar).EMSA—DNA binding experiments were performed using 50 ng of recombinant p53 proteins in a reaction mixture containing 5 ng of poly(dI-dC) (Amersham Biosciences) and 2 μg of bovine serum albumin in 50 mm Tris-HCl (pH 7.5), 0.1 mm EDTA, 1 mm dithiothreitol, 20% glycerol, and 50 mm NaCl. After a 20-min preincubation at room temperature 20,000 cpm of the labeled DNA probe was added, and the incubation was continued for an additional 25 min. Samples were loaded onto a 4% native polyacrylamide gel and separated by electrophoresis in 10 mm Tris-HCl (pH 7.8), 0.2 mm EDTA, 1.25 mm sodium acetate, and 8 mm acetic acid at 200 V for 2.5 h at room temperature. After electrophoresis gels were dried and analyzed by autoradiography.DNA Binding Assay Using FCS—A confocal microscope (ConfoCor; EVOTEC BioSystems and Carl Zeiss, Germany) was used for FCS studies. An attenuated (to about 800 microwatts) beam from an argon ion laser, wavelength 543 nm, was focused to a spot of ∼0.25-μm radius, resulting in a diffusion time of ∼60 μs for 6-carboxytetramethylrhodamine (TAMRA). The excitation intensity had generally been kept lower than or equal to a level characterized by about 15% amplitude of the triplet term of the autocorrelation function. Fluorescence emission was detected through a pinhole on the focal plane of the microscope using an avalanche photodiode detector SPCM-AQ 131 (EG&G) at 590 nm (bandwidth 35 nm).For the binding and the competition experiments human or mouse p53 was incubated with the TAMRA-labeled DNA for 15 min at 20 °C. In the case of competition titrations, the competitor was added together with the labeled DNA. In the case of activation of the sequence-specific DNA binding of p53 the protein was preincubated with PAb421 for 15 min at 20 °C before the addition of the DNA. All experiments were performed in 15-μl binding buffer (phosphate-buffered saline (pH 6.9), 0.05% Tween 20). Virtually identical results were obtained with human and mouse wild type p53. Binding data were fitted according to the standard hyperbolic binding model or according to the Hill equation (where mentioned), applying the fitting program Origin 6.0.RESULTSHigh Affinity Nonspecific DNA Binding Is a Specific Feature of Unmodified Wild Type p53 Protein—Sequence-specific DNA binding of p53 is commonly assessed by EMSA in the presence of competitor DNA to inhibit nonspecific DNA interactions. Depending on the kind of competitor DNA, its effects on sequence-specific DNA binding may greatly vary (10Anderson M.E. Woelker B. Reed M. Wang P. Tegtmeyer P. Mol. Cell. Biol. 1997; 17: 6255-6264Crossref PubMed Scopus (104) Google Scholar). Nonspecific DNA binding of p53 thus may represent an important parameter influencing sequence-specific DNA binding. We first analyzed by EMSA sequence-specific DNA binding and nonspecific DNA binding of p53 in the absence or presence of the antibody PAb421 (“unmodified” and “PAb421-modified” p53, respectively). PAb421 recognizes an epitope within the p53 C terminus (21Stephen C.W. Helminen P. Lane D.P. J. Mol. Biol. 1995; 248: 58-78Crossref PubMed Scopus (183) Google Scholar), and its binding is thought to mimic the binding of cellular proteins or posttranslational modifications that activate p53 for sequence-specific DNA binding. Unmodified wild type p53 bound with similar apparent affinities to both specific and unspecific DNA (DNAspec and DNAunspec, respectively) in the absence of competitor DNA (Fig. 1, A and B, lanes 2, complex A and A′, respectively). The addition of PAb421 strongly enhanced binding to DNAspec (Fig. 1A, lane 3, complex B′), in accordance with the proposed ability of PAb421 to activate sequence-specific DNA binding (7Hupp T.R. Lane D.P. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 195-206Crossref PubMed Scopus (106) Google Scholar). With DNAunspec, PAb421 only supershifted the complex p53·DNAunspec (Fig. 1B, lane 3, complex B) but did not enhance its formation.To estimate the impact of unspecific competitor DNA on sequence-specific DNA binding, we analyzed the effects of poly(dI-dC) on DNA binding of p53. Depending on whether or not p53 was modified by PAb421, poly(dI-dC) competed with specific and unspecific DNA with a different dose dependence. Although already the presence of 10 ng poly(dI-dC) almost completely abolished binding of PAb421-modified p53 to DNAunspec (Fig. 1B, lane 7, complex B), complete inhibition of binding of unmodified p53 to DNAunspec required much higher amounts (100 ng) of poly(dI-dC) (Fig. 1B, lane 11, complex A). The data suggest that PAb421-modified p53 binds weaker to unspecific DNA than unmodified p53. A reversed pattern was observed with DNAspec, as poly(dI-dC) much more efficiently competed out binding of unmodified p53 compared with binding of PAb421-modified p53 to DNAspec (Fig. 1A, lanes 2–17, complex A′ or B′, respectively).The results indicate that unmodified and PAb421-modified p53 differ in their binding to DNAspec and to DNAunspec. Whereas unmodified p53 binds DNAunspec and DNAspec equally well, the binding affinity of PAb421-modified p53 is shifted toward DNAspec. The weaker binding of PAb421-modified p53 to DNAunspec compared with unmodified p53 suggests that the p53 C terminus is involved in the high affinity interaction of unmodified p53 with DNAunspec. The conclusion is supported by the finding that nonspecific DNA binding of p53 was completely abolished by deletion of the C-terminal regulatory domain (CRD), as the deletion mutant p53-(1–360) lacking the CRD strongly bound to DNAspec (Fig. 1C, lanes 2–9) but completely failed to bind DNAunspec (Fig. 1D, lanes 2–9) even in the absence of competitor DNA (Fig. 1D, lane 2). Thus, high affinity nonspecific DNA binding of unmodified p53 requires the CRD.Importantly, mutant p53 proteins R248P and G245S not only were unable to bind DNAspec but also did not bind DNAunspec regardless of whether poly(dI-dC) was present or not (EMSA data not shown). Thus, the ability to bind unspecific double-stranded DNA with high affinity is an intrinsic biochemical property of wild type p53 that correlates with its potential to bind DNA sequence-specifically.Unmodified Wild Type p53 Does Not Discriminate between Specific and Unspecific DNA—To quantitatively analyze p53-DNA interactions in solution, we next examined by FCS the effects of PAb421 on DNA binding of wild type p53. Two types of TAMRA-labeled double-stranded DNA oligonucleotides were used, DNAspec, containing a p53 specific binding site, or DNAunspec, lacking such a sequence (see Table I for oligonucleotides used in FCS). The same DNAspec oligonucleotide had been used in the initial study describing the phenomenon of p53 latency and activation (8Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (859) Google Scholar). We first analyzed DNA binding of unmodified purified wild type p53 in the absence of poly(dI-dC). Binding titrations were performed at a constant concentration of TAMRA-DNA and increasing p53 concentration. Very similar dissociation constants (K D) of 17.7 ± 2.1 and 18.5 ± 2.2 nm were determined for the binding of tetrameric unmodified wild type p53 to TAMRA-DNAspec and to TAMRA-DNAunspec, respectively (Fig. 2A and Table II).Table IOligonucleotides (ODNs) used for binding analysis of p53ODNSequence (from 5′ to 3′)26-mer TAMRA-DNAspecTAMRA-AGCTTAGACATGCCTAGACATGCCTADNAspecAGCTTAGACATGCCTAGACATGCCTA35-mer DNAunspecTATGTCTAAGGGACCTGCGGTTGGCATTGATCTTG26-mer TAMRA-DNAunspecTAMRA-TATGTCTAAGGGACCTGCGGTTGGCA Open table in a new tab Fig. 2Characterization of DNA binding specificity of unmodified wild type p53 by FCS. A, protein titration experiments were performed to determine the binding affinity of wild type (wt) p53 and of mutant p53 R248P to DNAspec and DNAunspec using FCS. Titrations were performed at a constant concentration of TAMRA-labeled DNA (1 nm). The concentration of tetrameric p53 was varied. ▪, p53 wild type + TAMRA-DNAspec; •, p53 wild type + 26-mer TAMRA-DNAunspec; ▴, p53 R248P + TAMRA-DNAspec. B, displacement of TAMRA-DNAspec bound to unmodified wild type p53 with unlabeled DNAspec. The concentrations of tetrameric p53 (20 nm) and TAMRA-DNAspec (1 nm) were constant. The concentration of unlabeled DNAspec was varied. C, displacement of TAMRA-DNAspec bound to non-modified wild type p53 with unlabeled DNAunspec. The concentrations of tetrameric p53 (20 nm) and of TAMRA-DNAspec (1 nm) were constant. The concentration of unlabeled DNAunspec was varied.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIK D and K I values of wild type p53 interactions with DNADNABinding affinity (standard hyperbolic fitting)Binding affinity and Hill coefficient (Hill equationaHill equation: y = n × [p53]n/(KD + [p53]n) where y is the degree of DNA bound to p53, and n is the number of binding sites of p53 on DNA.)Location of resultspoly (dI-dC)PAb421nmTAMRA-DNAspecKD = 17.7 ± 2.1K D = 27.9 ± 9.1 nmFig. 2A--n = 1.3 ± 0.1TAMRA-DNAspecNot detectableFig. 5+-26-mer TAMRA-DNAunspecKD = 18.5 ± 2.2KD = 30.2 ± 8.7 nmFig. 2A--n = 1.3 ± 0.1DNAspecKI = 19.2 ± 6.0Fig. 2B--35-mer DNAunspecKI = 21.1 ± 4.5Fig. 2C--TAMRA-DNAspecKD = 1.1 ± 0.2KD = 1.0 ± 0.2 nmFig. 3A-+n = 1.4 ± 0.1TAMRA-DNAspecKD = 2.9 ± 0.5Fig. 5++26-mer TAMRA-DNAunspecKD = 169 ± 17KD = 627 ± 170 nmFig. 3A-+n = 1.3 ± 0.1DNAspecKI = 0.7 ± 0.4Fig. 3B-+35-mer DNAunspecKI = 2700 ± 1300Fig. 3C-+a Hill equation: y = n × [p53]n/(KD + [p53]n) where y is the degree of DNA bound to p53, and n is the number of binding sites of p53 on DNA. Open table in a new tab The minimal size of p53-specific binding sites corresponds to 20 base pairs and accommodates a single p53 tetramer (1Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2129) Google Scholar, 4Kern S.E. Kinzler K.W. Bruskin A. Jarosz D. Friedman P. Prives C. Vogelstein B. Science. 1991; 252: 1708-1711Crossref PubMed Scopus (934) Google Scholar). Although the oligonucleotides used here were only slightly larger (26 base pairs; see Table I), we still considered the possibility that more than one p53 tetramer could bind to a single oligonucleotide molecule. Such a binding behavior would influence the interpretation of the binding data shown above. To analyze the binding mode of p53 to DNA with respect to stoichiometry and cooperativity, we alternatively fitted the binding data applying the Hill equation y = n × [p53]n/(K D + [p53]n), where y is the portion of DNA bound to p53, and n is the number of p53 binding sites on the DNA (see Table II). The fitting results clearly show that for both, specific and unspecific DNA, the binding stoichiometry is 1:1 for the interaction partners and that no cooperativity is observed. In accordance with our EMSA experiments, mutant p53 R248P did not bind to either DNA, indicating that high affinity unspecific binding to linear DNA is a specific property of wild type p53 (Fig. 2A).The binding of unmodified wild type p53 to both DNAspec and to DNAunspec with similar high affinity suggested that such p53 was not able to discriminate between specific and unspecific DNA. The conclusion was corroborated by competition experiments which showed that TAMRA-DNAspec bound by unmodified p53 was displaced from the p53·DNA complex by unlabeled DNAspec and DNAunspec with comparable efficiencies (K I = 19.2 ± 6.0 and 21.1 ± 4.5 nm, respectively, Fig. 2B and C, Table II). The results of our FCS experiments are in accordance with our EMSA data and show that unmodified wild type p53 does not discriminate between sequence-specific and unspecific DNA and binds with high affinity to double-stranded linear DNA independent of the presence of a cognate binding motif.PAb421 Increases the Binding Affinity of Wild Type p53 for DNA spec While Concomitantly Decreasing the Binding Affinity for DNAunspec—We next analyzed the effects of PAb421 on the binding of p53 to DNAspec and to DNAunspec. Fig. 3A and Table II show that the affinity of PAb421-modified p53 for TAMRA-DNAspec increased only moderately, i.e. by a factor of ∼15 (K D = 1.1 ± 0.2 nm). In accordance, the K I for DNAspec also dropped to 0.7 ± 0.4 nm (Fig. 3B, Table II). In contrast to the increase in affinity for TAMRA-DNAspec by PAb421, the affinity of PAb421-modified p53 for TAMRA-DNAunspec was greatly reduced (K D = 169 ± 17 nm, Fig. 3A and Table II). The net outcome of these adverse effects of PAb421 on the respective p53-DNA interactions is a drastic (150-fold) difference in the affinity of PAb421-modified p53 for DNAspec compared with DNAunspec. Competition experiments using the 35-mer DNAunspec, used in the initial study describing the latency phenomenon (8Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (859) Google Scholar), showed an even larger (∼3,000-fold) difference in the affinity of PAb421-modified p53 to unspecific DNA. The 35-mer DNAunspec was only able to effectively displace TAMRA-DNAspec from the complex with PAb421-modified p53 with a KI of 2700.0 ± 1300 nm (Fig. 3C, Table II). The results imply that modification of p53 by PAb421 leads to a significant reduction of its binding affinity for unspecific DNA, with the degree of reduction possibly varying with DNA length. Further analysis of the binding data using the Hill equation showed that PAb421 did not influence stoichiometry or cooperativity of binding (Table II). As expected, mutant p53 R248P did not bind to DNAspec even in the presence of PAb421 (Fig. 3A).Fig. 3DNA binding specificity of PAb421-modified wild type p53 analyzed by FCS. A, protein titration experiments were performed to determine the binding affinity of wild type (wt) p53 to DNAspec and DNAunspec after the addition of PAb421 (250 nm). Titrations were performed at a constant concentration of TAMRA-labeled DNA (1 nm). The concentration of tetrameric p53 was varied. ▪, wild type p53 + TAMRA-DNAspec; ▴, wild type p53 + 26-mer TAMRA-DNAunspec; •, mutant p53 R248P + TAMRA-DNAspec. B, displacement of TAMRA-DNAspec bound to PAb421-modified wild type p53 with DNAspec. The concentrations of tetrameric p53 (2 nm), of TAMRA-DNAspec (1 nm), and PAb421 (250 nm) were constant. The concentration of DNAspec was varied. C, displacement of TAMRA-DNAspec bound to PAb421-modified wild type p53 with DNAunspec. The concentrations of tetrameric p53 (2 nm), TAMRA-DNAspec (1 nm), and PAb421 (250 nm) were constant. The concentration of DNAunspec was varied.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Again, the results of the FCS analyses are concordant with our EMSA data (Fig. 1). The data suggest that PAb421 modulates the specificity of p53 DNA binding rather than increasing its binding affinity for DNAspec. We propose that PAb421 has a dual effect on p53, leading to sequence-specific recognition of DNAspec accompanied by a significant decrease in the affinity of p53 for DNAunspec.Fig. 4A demonstrates another important aspect of the activation of p53 DNA binding by PAb421. At low molar ratios of p53:DNAspec (2:1 nm in Fig. 4A), DNA binding by unmodified p53 was hardly detectable because the concentrations of the binding partners were much below the K D of ∼17.7 ± 2.1 nm (see Fig. 2A). Under such conditions, the addition of PAb421 strongly enhanced the fraction of DNAspec bound by p53. The enhancement, however, can be solely explained by the moderately higher affinity of PAb421-modified p53 to DNAspec (K D = 1.1 ± 0.2 nm) compared with that of unmodified p53 (K D = 17.7 ± 2.1 nm). Fig. 4A thus exemplifies that under certain conditions even a moderate difference in the binding affinity of p53 to a given DNA substrate can be relevant. In addition, Fig. 4A, as an important control, shows that PAb421 as such does not bind DNA. Furthermore, activation of p53 is specific for PAb421, because the addition of PAb1801, binding to an epitope in the p53 N terminus (22Leppard K. Totty N. Waterfield M. Harlow E. Jenkins J. Crawford L. EMBO J. 1983; 2: 1993-1999Crossref PubMed Scopus (25) Google Scholar), did not have an effect on p53 binding to DNAspec.Fig. 4Influence of p53 specific antibodies on p53 binding to DNAspec. A, enhancement of p53 DNAspec binding by monoclonal antibody PAb421 at a low p53·DNA ratio. The concentrations of tetrameric p53 (2 nm) and of TAMRA-DNAspec (1 nm) were constant. The concentration of PAb421 varied. ▪, p53 + TAMRA-DNAspec + PAb421; ▴, no p53 + TAMRA-DNAspec + PAb421. •, N-terminal PAb1801 was added to p53·DNAspec complexes in the same" @default.
• W1973291880 created "2016-06-24" @default.
• W1973291880 creator A5008229579 @default.
• W1973291880 creator A5017560239 @default.
• W1973291880 creator A5019233746 @default.
• W1973291880 creator A5020407174 @default.
• W1973291880 creator A5080432309 @default.
• W1973291880 creator A5091514112 @default.
• W1973291880 date "2003-08-01" @default.
• W1973291880 modified "2023-10-02" @default.
• W1973291880 title "Analysis of p53 “Latency” and “Activation” by Fluorescence Correlation Spectroscopy" @default.
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