p53 Latency
C-TERMINAL DOMAIN PREVENTS BINDING OF p53 CORE TO TARGET BUT NOT
TO NONSPECIFIC DNA SEQUENCES*
Tatiana
Yakovleva
,
Aladdin
Pramanik§,
Takashi
Kawasaki¶,
Koichi
Tan-No
,
Irina
Gileva
,
Heléne
Lindegren
,
Ülo
Langel**
,
Tomas J.
Ekström
,
Rudolf
Rigler§,
Lars
Terenius
, and
Georgy
Bakalkin
§§
From the
Experimental Alcohol and Drug Addiction
Research Section, Department of Clinical Neuroscience, the
§ Department of Medical Biochemistry and Biophysics, and the
¶ Microbiology and Tumor Biology Center, Karolinska Institute and
the ** Department of Neurochemistry and Neurotoxicology, Stockholm
University, Stockholm, Sweden
Received for publication, January 18, 2001, and in revised form, February 20, 2001
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ABSTRACT |
The p53 transcription factor is either latent or
activated through multi-site phosphorylation and acetylation of the
negative regulatory region in its C-terminal domain (CTD). How CTD
modifications activate p53 binding to target DNA sequences via its core
domain is still unknown. It has been proposed that nonmodified CTD
interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here
characterized the sequence and target specificity of p53 activation by
CTD fragments, interaction of activating peptides with p53 and target
DNA, and interactions of "latent" p53 with DNA by a band shift
assay and by fluorescence correlation spectroscopy. In addition to CTD
fragments, several long basic peptides activated p53 and also
transcription factor YY1. These peptides and CTD aggregated target DNA
but apparently did not interact with p53. The potency to aggregate DNA
correlated with the ability to activate p53, suggesting that p53 binds
to target sequences upon interactions with tightly packed DNA in
aggregates. Latent full-length p53 dissociated DNA aggregates via its
core and CTD, and this effect was potentiated by GTP. Latent p53 also
formed complexes via both its core and CTD with long nontarget DNA
molecules. Such p53-DNA interactions may occur if latent p53 binding to
DNA via CTD prevents the interaction of the core domain with target DNA
sites but not with nonspecific DNA sequences.
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INTRODUCTION |
The p53 tumor suppressor protein is a latent transcription factor
that is activated by various forms of cellular stress including DNA
damage. Activation of p53 binding to target DNA sequences and,
consequently, p53-dependent transcription is controlled by the negative regulatory region in the CTD of p53, which includes amino
acids 361-382. Post-translational modifications of amino acid residues
in this region through phosphorylation activate sequence-specific DNA
binding. Acetylation of several C-terminal lysines by p300/CBP/PCAF,
recruited through phosphorylation of the distant N-terminal serines,
critically regulates the site-specific DNA binding function of p53 (for
reviews see Refs. 1-3). p53 exists in vitro in a latent
form that cannot bind to p53-responsive DNA elements (4). Deletion of
the critical regulatory region in the 30-amino acid C-terminal domain
(CTD)1 results in p53
activation for sequence-specific DNA binding. Furthermore, binding of
the monoclonal antibody PAb421 to this region activates p53
sequence-specific DNA binding and triggers the transcriptional activity
of p53 in vivo (4).
The mechanism of the CTD-mediated inhibition of p53 sequence-specific
DNA binding is not understood. It has been proposed that by
intramolecular interaction with the p53 DNA-binding core, CTD
allosterically locks the p53 molecule in a state latent for binding to
DNA (4). In accordance with this model, CTD or CTD fragments including
p53(361-382) can displace CTD from its binding site within the central
domain, resulting in a change in p53 conformation and activation of p53
binding to DNA (4). The CTD fragments can bind to the isolated core
domain in vitro (5, 6). Activation of latent p53 by peptides
derived from CTD has been reported to be sequence-specific because
neither mutant p53(361-382) peptides nor peptides derived from the
N-terminal p53 domain activate p53 or demonstrate only weak activity
(4, 5, 7). CTD also shows target specificity because it did not
influence the interaction of SRF and GAL4-VP16 proteins with DNA (8).
The overlap of the regulatory region in CTD, which is rich in basic
amino acids, with the second DNA-binding site in the p53 molecule is
the key feature of the steric model (9, 10). This model postulates that
p53 binds to genomic DNA via CTD, and this binding prevents the
interaction of the core domain with target sequences in gene promoters.
The steric model is supported by the fact that long nonspecific
DNA molecules inhibit sequence-specific binding of the full-length p53
to DNA but shows no effect on p53 with deletion of the CTD regulatory
sequence (9, 10).
To gain insight into the mechanism of p53 latency, the sequence and
target specificity of p53 activation by CTD fragments and the
interactions of activating peptides with p53 and DNA were characterized. A conventional band shift assay was used to characterize p53 binding to DNA, whereas fluorescence correlation spectroscopy (FCS)
was applied to follow the interactions of activating peptides with p53
and DNA. The high sensitivity of FCS (Fig.
1 and Refs. 11-15) allows analysis of
such interactions and their nature even if they are weak in a reaction
mixture. DNA molecules unexpectedly formed aggregates in the presence
of the CTD fragment and other activating peptides. Our findings suggest
that p53 is activated for binding to target DNA sites when it interacts
with juxtaposed DNA molecules in DNA aggregates via both its core and
CTD. Thus, CTD, when it binds to DNA, may prevent the interaction of
the core domain of the full-length p53 with target but not with
nontarget DNA sequences. This hypothesis seems to unify the previously
postulated steric and allosteric models.

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Fig. 1.
Fluorescence correlation spectroscopy
setup. The laser beam from an argon ion laser is sharply focused
via a dichroic mirror and a lens to form a tiny confocal volume element
of 0.2 fl. The laser beam is projected from below into a eight-well
Nunc chamber containing rhodamine-labeled compounds (see
magnification). After excitation of the labeled compounds, emitted
light is transmitted via the dichroic mirror, a bandpass filter, and a
pinhole to a photodetector. The volume element is positioned into
reaction medium. The dimensions of the laser beam focus and the pinhole
together define the confocal volume element from which fluorescent
light is collected. The photodetector operates in a photon counting
mode, responding with an electrical pulse to each detected photon. The
detector signal (electrical pulse) is fed into a digital signal
correlator that calculates online in real time the autocorrelation
function of the detected intensity fluctuations. G( ),
autocorrelation function. D1 and D2,
diffusion times for free and bound fluorophore, respectively;
D2 D1.
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EXPERIMENTAL PROCEDURES |
Plasmids, Proteins, and Peptides--
The plasmid encoding human
wild type p53, GST-human wild type p53 fusion protein, the deletion
fusion proteins GST-p53(1-100), GST-p53(99-307), GST-p53(320-393),
and GST-p53
30, the mutant fusion proteins GST-p53His273 and
GST-p53Trp248 have been described elsewhere (5, 16-18). The PG and MG
fragments with 13 copies of consensus and 15 copies of mutant sites,
respectively, were obtained by digestion of the PG-CAT and MG-CAT
plasmids with HindIII/EcoRI. The RD fragment was
cut from the pRC/CMV-Dyn plasmid. Plasmid DNA and oligonucleotides were
purified by the Qiagen kit (Hilden, Germany) and polyacrylamide-urea
gel electrophoresis, respectively; their concentrations were determined
from their absorbance at 260 nm, and their extinction coefficients were
calculated from nucleotide composition.
p53 proteins were produced in Escherichia coli, purified as
outlined earlier, and quantitated by Coomassie Blue or silver staining
following SDS-polyacrylamide gel electrophoresis with bovine serum
albumin as a standard (17, 18). The basic 32-amino acid fragment of
human prodynorphin (prodynorphin 207-238 or big dynorphin (BD),
YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVT), dynorphins A (BD1-17) and B
(BD20-32) were obtained from Phoenix Pharmaceuticals Inc. (Mountain
View, CA), and penta-L-lysine and poly-L-lysine were purchased from Bachem (Bubendorf, Switzerland). Eight CTD fragments (22-mers) spanning CTD residues 337-393 with a 14-residue overlap including p53(361-382) (GSRAHSSHLKSKKGQSTRHKK) were described earlier (18).
DNA Binding Assays--
Binding of p53 to specific sequences was
performed as described earlier (4, 18) in the presence of 2 mM MgCl2. 1 nM
32P-labeled 30-mer BC oligonucleotide containing
five adjacent copies of p53 consensus pentamer binding site and GST-p53
protein (35 nM of monomer) were incubated in the absence or
in the presence of 25 nM of nonspecific competitor
poly(dI-dC). p53 proteins binding to long 32P-end-labeled
DNA was described earlier (17). AP-1, NF-
B, YY1, and protein Ku were
assayed in nuclear extracts from SH-SY5Y cells and YY1 with purified
protein as described earlier (19, 20).
Fluorescent Compounds--
BD with the C-terminal Cys-amide
extension was synthesized and labeled with
tetramethylrhodamine-5-iodoacetamide (Molecular Probes Europe BV)
according to the manufacturer's instructions. The Rh-BD was isolated
on an analytical Nucleosil 120-3 C18 reverse phase HPLC
column (Macherey-Nagel) and lyophilized, and the powder was weighed and
used for preparation of solutions. The identity of the purified labeled
peptide was confirmed by plasma desorption mass spectrometry. The
purity of the peptide was > 90%.
5-Carboxytetramethylrhodamine (Rh')-labeled ((+)-strands) and unlabeled
((
)-strands), HPLC-purified DNA 50-mer oligonucleotides were
purchased from TIB-MOLBIOL (Berlin, Germany). The oligonucleotides represent the wild type and mutant BC 30-mer oligonucleotides, extended
from 5' and 3' ends, and contain six adjacent consensus pentamer wild
type or mutant (mutant nucleotides are shown in italic type) p53
binding sites (underlined; the sequences for the (+)-strands are
shown):
Rh'-5'-AGTCGTCGACCGGGCATGTCCGGGCATGTCCGGGCATGTCCCGTACTAGG-3' (ss-Rh'-SO) and
Rh'-5'-AGTCGTCGACCGCGTACTGTGGGCGATCGGCGACACGTCTCCGTACTAGG-3' (ss-Rh'-NO), respectively. Concentrations of fluorescent
single-stranded oligonucleotides were determined from their
absorbance at 260 nm using the extinction coefficients calculated from
nucleotide composition after subtraction of the absorbance of
5-carboxytetramethylrhodamine at this wavelength. Contribution of
5-carboxytetramethylrhodamine to the absorbance at 260 nm was
calculated from the spectrum of this dye and from the absorbance of
fluorescent oligonucleotides at 546 nm.
Concentrations of fluorescent peptide and oligonucleotides, as well as
the presence of free dye were verified by measuring the rhodamine
absorbance at 546 nm and by FCS with 1 nM rhodamine standard solution prepared by weighing. At 1 nM
concentration of a standard rhodamine solution and solutions of
fluorescent peptides and oligonucleotides, 0.2 fl confocal
volume of measurement contains in average one molecule of fluorescent
dye that was detected by FCS. Free dye in solutions did not exceed 10%
of fluorescently labeled compounds.
FCS Instrumentation--
FCS was performed with confocal
illumination of a volume element of 0.2 fl in a ConfoCor® instrument
(Carl Zeiss-Evotec, Jena, Germany; Fig. 1) as described previously
(21). As focusing optics a Zeiss Neofluar 40× numerical aperture 1.2 objective for water immersion was used in an epiillumination setup.
Separation of exciting from emitted radiation was achieved by dichroic
(Omega 540 DRL PO2; Omega Optical, Blattleboro, VT) and
bandpass (Omega 565 DR 50) filters. The Rh-BD and Rh'-oligonucleotides
were excited with the 514.5-nm line of an argon laser. The intensity
fluctuations were detected by an avalanche photodiode (SPCM 200; EG & G, Quebec, Canada) and processed with a digital correlator (ALV 5000, ALV, Langen, Germany).
FCS Data Evaluation--
The intensity autocorrelation function
G(t) is an average of the product between the
intensity and its time shifted version (Equation 1).
or
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(Eq. 1)
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Correlation of the observed fluorescence intensity fluctuations
I(t) with fluorescence intensity fluctuations
at time t +
yields the normalized intensity
autocorrelation function G(
).
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(Eq. 2)
|
where the brackets describe the time average and
<I> describes the mean fluorescence intensity (21,
22).
In our experiments fluorescence intensity fluctuations
I(t) occurring in a volume element of 0.2 fl
with half-axes
= 0.25 µm and z = 1.25 µm
are correlated (21, 22), and for calculation of parameters of the
autocorrelation function G(
) the nonlinear least square
minimization is used (23). The intensity autocorrelation function for
three-dimensional diffusion (13) of the unbound Rh-BD or Rh'-SO and
bound Rh-BD or Rh'-SO is given by the following equation.
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(Eq. 3)
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N is the number of molecules,
DB =
2/4DB is
the diffusion time for bound Rh-BD or Rh'-SO;
DF =
2/4DF is the diffusion time for
unbound Rh-BD or Rh'-SO; y is the fraction of bound Rh-BD or
Rh'-SO diffusing with
DB; (1
y) is the fraction of unbound Rh-BD or Rh'-SO diffusing with
DF.
FCS Experiments--
Fluorescent probes Rh-BD, Rh'-SO, or Rh'-NO
were incubated at 2 nM concentrations in the absence or the
presence of p53 proteins, peptides, or other compounds in binding
buffer (20 mM HEPES, pH 7.4, 50 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 1 mM dithiothreitol, 20% glycerol, and 0.05% Triton X-100)
used for p53-DNA binding studies at 20 °C, and droplets (10 µl) of
the samples were analyzed by FCS for 1 up to 3 min. Bovine serum
albumin (1 µg/µl) was included when Rh'-SO and Rh'-NO
oligonucleotides were used as fluorescent probes. Measurements were
performed in triplicate 2-5, 10-20, and 30-40 min after the
initiation of the reaction. Practically identical results were obtained
at these time points.
Antibodies--
Anti-YY1 C20 antibody with blocking peptide and
anti-p50 antibody were obtained from Santa Cruz Biotechnology (San
Diego, CA), anti-p53 PAb421 antibody was from Oncogene Science Inc.
(Unlondale, NY).
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RESULTS |
Activation of p53 Binding to Specific DNA Sequences by Basic
Peptides--
Sequence specificity of peptide effects on p53 binding
to DNA was studied in a band shift assay. As previously described (18), the GST-wild type p53 protein remained latent in the absence of any DNA
competitor (Fig. 2, lane 1).
p53 binding to the consensus p53-binding site, BC, was activated by
PAb421 antibody (lane 2), and by the p53(361-382) fragment
(lane 3). p53(361-382) is a basic peptide, which suggests
that other basic peptides may also activate p53. Three model basic
peptides with irregular alternation of basic residues, the 32-amino
acid fragment of prodynorphin (human prodynorphin 207-238 or BD) and
two of its constituent fragments, dynorphin A (17 amino acids) and B
(13 amino acids), as well as penta-L-lysine and
poly-L-lysine with a size up to 5 kDa, were tested for
effects on p53 binding to DNA. Both BD and poly-L-lysine strongly activated p53 (lanes 4, 15, and
16), whereas dynorphins A and B produced practically no
activation (lanes 5 and 6). No activation was
observed with penta-L-lysine at ~30-fold higher molar
concentration (lanes 12 and 13) or a mixture of
dynorphins A and B at a concentration equimolar with BD (lane
7). No stable complexes were seen when radiolabeled DNA was
incubated with these peptides alone (lane 11 and data not
shown). Complex formation activated by BD was inhibited by unlabeled BC
oligonucleotide (lane 9) but not by unlabeled
oligonucleotide MN lacking p53 binding site (lane 10; Ref.
18), demonstrating that the peptide activated sequence-specific
interaction with DNA. p53-labeled BC complex formation was activated by
basic peptides in the absence of any competitor DNA (lanes 3 and 4) or in the presence of poly(dI-dC) (lanes
8, 10, 15, and 16).

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Fig. 2.
Effects of basic peptides on
sequence-specific p53 DNA binding. A band shift assay of GST-wild
type p53 protein (lanes 1-10 and 12-16; 35 nM of p53 monomer) incubated with the monoclonal antibody
PAb421 (70 nM), the p53(361-382) (lane F; 2.6 µM), BD (lanes 4 and 8-10; 2.6 µM), dynorphin A (lane A, 2.6 µM), dynorphin B (lane B, 2.6 µM), a mixture of dynorphins A (2.6 µM) and
B (2.6 µM), penta-L-lysine (lanes
5L; lane c, 17 µM; lane d, 34 µM), poly-L-lysine (lanes PL;
lane a, 0.6 µM; lane b, 1.2 µM; lane c, 2.5 µM) and labeled
30-mer BC oligonucleotide containing p53 consensus binding site.
BD-activated p53-DNA-binding was inhibited by nonlabeled BC (lane
S; 25 nM) but not by MN oligonucleotide (lane
N; 25 nM). BD (2.6 µM) itself did not
produced a stable complex with labeled BC (lane 11). Assay
was performed in the presence (lanes 8-10 and
12-16) or in the absence (lanes 1-7 and
11) of poly(dI-dC).
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Activation of YY1 by Basic Peptides and p53--
To test whether
p53(361-382) and BD can target transcription factors other than p53,
their effects on the DNA binding activity of AP-1, NF-
B, YY1, and
protein Ku were studied in nuclear extracts of SH-SY5Y cells. Only the
DNA binding activity of YY1, a multifunctional transcription regulator,
was activated by the two peptides (data not shown). Experiments were
repeated with affinity purified YY1, and DNA binding was strongly
(10-50-fold) activated by p53(361-382) and BD (Fig.
3A, lines 1-6)
with half-maximum effects observed at 5-10 nM
concentrations (data not shown). Activated YY1 retained its sequence
specificity because an oligonucleotide with wild type but not mutant
binding site inhibited binding (lanes 7-9). C20 anti-YY1
antibodies supershifted the protein-DNA complex activated by
p53(361-382), showing that YY1 is present in the complex (lane 13), whereas other antibodies or C20 antibodies preincubated with a blocking peptide (lanes 11 and 12) failed to
inhibit YY1 activation.

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Fig. 3.
Stimulation of the YY1 DNA binding activity
by basic peptides, p53 domains, and p53 in a band shift assay.
Reaction mixture contained 0.15 nM of affinity purified
YY1, 32P-labeled LINE oligonucleotide with YY1-binding
site, and 20 nM poly(dI-dC). A, effects of
p53(361-382) (lanes F, lane a, 0.9 µM; lane b, 2.2 µM; lanes
7-13, 2.2 µM) and BD (lanes BD,
lane a, 0.5 µM; lane b, 1.3 µM) on the YY1 DNA binding. Wild type (wt) and
mutant (m) YY1 consensus binding site oligonucleotides (20 nM) were used as competitors (5). Anti-YY1 antibodies C20
(lane 13) but not C20 or control anti-p50-antibodies
(lane a) preincubated with C20 blocking peptide (bp;
lanes 11 and 12) supershifted YY1-DNA complexes.
B, effects of the GST protein alone (36 nM),
GST-p53 deletion mutant proteins representing NTD (residues 1-100;
lane N; 25 nM) and the core domain (residues
99-307; lane M; 19 nM), the deletion mutant
lacking the 30 C-terminal amino acids (lane C;
14 nM), the GST-wild type p53 (0.7 nM), GST-CTD
(residues 320-393; 2.7 nM), and as well as GST-mutant p53
proteins p53Arg273 (m273; 3.5 nM) and p53Trp248 (m248; 3.5 nM) on YY1-DNA binding. C, effects of eight
peptides (22-mers; 8 µM) spanning CTD residues 337-393
with a 14-residue overlap on YY1 binding to DNA; lane f
corresponds to p53(361-382).
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To determine whether the basic segment within the p53 protein is able
to activate YY1, the effects of the full-length p53 and p53 domains on
the YY1 DNA binding activity were compared. GST-wild type p53 at a low
concentration (0.7 nM) substantially (20-50-fold)
stimulated specific DNA binding of purified YY1 (Fig. 3B,
lane 6). GST-CTD, GST-p53His273, and GST-p53Trp248 fusion proteins also enhanced DNA binding activity of YY1 (lanes 8,
10, and 11). The lower band apparently
represented the truncated YY1-DNA complex. Myoglobin, casein, RNase,
polynucleotide kinase, bovine serum albumin, GST-retinoblastoma, and
GST-EBNA5 fusion proteins used as controls at 15 nM
concentration failed to modify YY1-DNA binding (data not shown). GST
protein alone, GST-p53-N-terminal domain (NTD), and core domains and
the GST-p53 deletion mutant lacking the 30 C-terminal amino acids
failed to activate YY1 (lanes 2-5). Thus, activation is
associated with CTD. To map the sequence of activation in closer
details, eight partly overlapping 22-mer peptides spanning most of the
C-terminal domain (residues 337-393) were examined. Only the most
basic CTD fragment, p53(361-382) retained the ability to strongly
activate YY1 (Fig. 3C, lane 6). The basic CTD
segment in latent p53 appears to be available for intermolecular
interaction with YY1 or YY1-target oligonucleotides but, paradoxically,
does not activate p53 itself (Fig. 2, lane 1).
No Binding of the Activating Basic Peptide BD to p53--
Basic
peptides may activate p53 by interacting with the p53 protein, with
target DNA or with both molecules. Multiple peptide-protein and
peptide-DNA interactions including formation of complexes of weakly
interacting molecules at their nanomolar concentrations cannot be
readily analyzed by conventional biochemical methods. FCS, a new highly
sensitive biophysical technique, allows the analysis of weak
interactions and intermolecular complex formation without preceding
separation of a fluorescently labeled probe from labeled complexes
(Fig. 1 and Refs. 11-15). In the first set of experiments,
interactions of the tetramethyl rhodamine-labeled basic peptide, BD
(Rh-BD) as a fluorescent probe with the p53 protein and target DNA were
studied by FCS. BD, but not p53(361-382), was chosen to distinguish
the interactions relevant for the p53 activation from those in which
p53(361-382) may be also involved as a fragment of the p53 molecule.
Rh-BD was incubated alone or with either GST-p53 or target
oligonucleotide ds-SO. Aliquots of the incubation mixture were analyzed
by FCS after 2-5, 10-20, and 30-40 min.
The fluctuations (changes) of the fluorescence intensity of
fluorophore-labeled BD molecules, excited by a focused laser beam, were
registered (Figs. 1 and 4A).
These fluctuations reflected a change in the number of Rh-BD molecules
in the volume of measurement and were expressed in kHz. At 2 nM concentration of fluorophore, an average of two
fluorescent molecules are present in the volume. The
fluorescence intensity is increased when new fluorescent molecules diffuse into the volume and decreased when some molecules diffuse out
of the volume. From the intensity autocorrelation function (Equations
1-3) of the fluorescence intensity fluctuations, the diffusion time of
molecules (
D) through the confocal volume is determined,
which allows calculations of the molecular weight. Formation of
complexes of fluorophore with other molecules in the reaction medium or
aggregation of the fluorophore molecules result in an increase of
molecular weight of fluorescent complexes and, consequently, their
diffusion time (
D). From the correlation function,
G(
) (Equation 3) diffusion times and proportions (weight factors, y) of different fluorescent components in the
reaction mixture were evaluated.

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Fig. 4.
Interaction of Rh-BD with p53 and DNA.
Rh-BD was incubated alone and in the presence of GST-p53 and 50-mer
ds-SO, which contained p53 binding site. Aliquots of the incubation
mixture were analyzed by FCS. A, fluorescence intensity
fluctuations for Rh-BD (2 nM) alone; B, Rh-BD
plus 1 nM of ds-SO. C, normalized
autocorrelation functions G( ) for: Rh-BD alone
(curve 1; diffusion time D = 0.175 ms), Rh-BD
plus 37 nM of GST-p53 (curve 2; D = 0.181, p53 monomer concentration is given), and Rh-BD plus 1 nM ds-SO (curve 3; D and
corresponding fraction y: D1 = 0.175 ms,
y1 = 0.35; D2 = 3899 ms,
y2 = 0.65). Curves 2 and 3 are completely overlapping. Data were recorded 10-20 min after the
initiation of the reaction.
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Fluorescence intensity fluctuations and autocorrelation functions of 2 nM Rh-BD alone and in the presence of GST-p53 fusion protein and DNA are presented in Fig. 4. Rh-BD (control) exhibited typical fluctuations (Fig. 4A) and a diffusion time
(
D) of 0.175 ms (Fig. 4C, curve
1). GST-p53 in the concentration range from 6 to 37 nM
(Fig. 4C, curve 2, and data not shown) did not
have any effect on the diffusion time of Rh-BD. Addition of 1 nM double-stranded specific (ds-SO; Fig. 4B) or
double-stranded nonspecific (data not shown) oligonucleotide containing
wild type or mutant p53 consensus sequences, respectively, as well as
0.24 nM of plasmid DNA (data not shown), to the reaction
mixture with Rh-BD resulted in an increase and a broadening of the
fluctuation peaks. The fluorescence intensity fluctuations exhibited
several peaks with strongly increased heights (Fig. 4B).
These peaks, with up to five times higher intensity than the base line,
are a clear representation of the Brownian motion of peptide-DNA
aggregates containing several tetramethyl rhodamine-labeled BD molecules.
The analysis of the autocorrelation of intensity fluctuations showed
the presence of two fluorescent components in the reaction mixture
characterized by diffusion times of
D1 = 0.175 ms and
D2 = 3899 ms, respectively, and corresponding weight
factors (fractions) of y1 = 0.35 and
y2 = 0.65 (Fig. 4C, curve
3). The appearance of the second component with longer diffusion
time reflected the formation of large Rh-BD-DNA aggregates. From the diffusion times 0.2 and 4 s, the hydrodynamic radii of equivalent spheres were calculated (14) according to the Stoke-Einstein equation
and were found to be 4 and 80 µm, respectively. The three-dimensional structure of aggregates is unclear, and, therefore, calculations assuming other shapes than a sphere were not performed. Practically identical results were obtained at different time points after the
initiation of the reaction. Thus, at nanomolar concentrations of basic
peptide, protein, and DNA, Rh-BD did not interact with p53 but
aggregated DNA. Low affinity binding of BD to p53 cannot, however, be
ruled out.
Aggregation of DNA by CTD and Basic Peptides--
In the second
set of experiments, interactions of activating peptides including the
CTD fragment p53(361-382), and proteins including p53 domains and
"latent" p53, with fluorescent target oligonucleotides were studied
by FCS (Fig. 5). Fluorescent Rh'-SO and
Rh'-NO oligonucleotides, which contain wild type and mutant p53 target
sites, respectively, were used as labeled probes. Fluorescence intensity fluctuations and autocorrelation function of Rh'-SO are shown
in Fig. 5 (A and D, curve 1),
respectively. Rh'-SO or Rh'-NO were incubated with peptides and p53
proteins, fluorescence intensity fluctuations were registered, and
autocorrelation functions were evaluated. GST-p53 (37-74
nM), GST-p53-N-terminal domain (25 nM), and GST
protein alone (25 nM) did not change fluorescence intensity
fluctuations (data not shown) and autocorrelation functions of 2 nM Rh'-SO (Fig. 5D, curves 1-4) or
Rh'-NO (data not shown). Interactions of GST-CTD (7-35 nM;
Fig. 5B), GST-core domain (24 nM; data not
shown), and BD (3 µM; Fig. 5C) with Rh'-SO
(Fig. 5, B and C) and Rh'-NO (data not shown)
resulted in an increase and a broadening of the intensity fluctuation
peaks. Correlation analysis of the intensity fluctuations showed a
diffusion process with two fluorescent components characterized by
diffusion times of
D1 = 0.692 ms and
D2 = 3567-3890 ms, respectively, and corresponding weight factors
(fractions) of y1 = 0.3-0.4 and
y2 = 0.6-0.7 (Fig. 5D, curves
5-7). The first component with relatively short diffusion time of
D1 = 0.692 ms corresponded to the free molecules of
Rh'-SO, whereas the second component with longer diffusion time of
D2 = 3567-3890 ms represented large Rh'-SO-protein or
peptide aggregates because diffusion coefficient relates to the size of
a molecule or molecular complex. Thus, CTD, p53 core and BD formed
large aggregates with DNA at nanomolar concentrations of DNA and
proteins and at micromolar concentrations of the peptide. Plasmid DNA
(1.9 nM) completely inhibited interactions of CTD and core
domains with fluorescent oligonucleotides (Fig. 5D, compare
curves 5 and 6 with curves 8 and
9), demonstrating that DNA-binding sites of both domains
were involved in DNA aggregation. Practically identical results were
obtained at different time points after the initiation of the reaction.
KCl at 300 mM concentration inhibited the aggregation (data
not shown), indicating an electrostatic character of peptide-DNA interactions.

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Fig. 5.
Interaction of p53, p53 domains, and BD with
Rh'-SO, which contains p53-binding sites. Rh'-SO was incubated
alone and in the presence or absence of GST-p53, p53 domains, and basic
peptide BD. The incubation mixture was analyzed by FCS. A,
fluorescence intensity fluctuations for Rh'-SO (2 nM)
alone. B, Rh'-SO plus 35 nM GST-CTD.
C, Rh'-SO plus 3 µM BD. D,
normalized autocorrelation functions G( ) for: Rh'-SO
alone (curve 1; D = 0.692 ms), Rh'-SO plus 37 nM GST-p53 (curve 2; D = 0.701 ms), Rh'-SO plus 25 nM GST-p53-N-terminal domain
(curve 3; D = 0.717 ms), Rh'-SO plus 37 nM GST (curve 4; D = 0.720 ms),
Rh'-SO plus 35 nM GST-CTD (curve 5;
D = 3890 ms), Rh'-SO plus 24 nM GST-core
domain (curve 6; D = 3567 ms), Rh'-SO plus 3 µM BD (curve 7; D = 3809 ms),
Rh'-SO plus 35 nM GST-CTD + 1.9 nM plasmid DNA
(curve 8; D = 0.697 ms), and Rh'-SO plus 24 nM GST-core domain + 1.9 nM plasmid DNA
(curve 9; D = 0.714 ms). Curves
1-4, 8, and 9 are completely overlapping.
Concentrations of protein monomeric forms are given. Data were recorded
10-20 min after the initiation of the reaction.
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|
Interactions of other basic peptides poly-L-lysine and
p53(361-383) with fluorescent oligonucleotides also increased
diffusion time of a fraction of fluorophore (y = 0.3-0.7) (summarized in Table I).
Poly-L-lysine aggregated DNA at 3- and 20-fold lower molar
concentrations compared with BD and p53(361-383), respectively, whereas penta-L-lysine was not effective at 240-fold higher
molar concentration. The aggregation potential of basic peptides
correlated with their ability to activate latent p53 for
sequence-specific DNA binding in a band shift assay (Fig. 2 and Table
I).
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Table I
Aggregation of Rh'-SO by basic peptides
/+ and +, aggregation with the weight factors from 0.2 to 0.3 and
from 0.3 to 0.7, respectively; , no aggregation. ND, Not determined.
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Inhibition of DNA Aggregation by p53--
In the third set of
experiments, effects of activating peptides on p53-DNA interactions
were studied by FCS. Rh'-SO containing p53 target sequences was used as
a fluorescent probe. BD alone aggregated Rh'-SO (Figs.
6, A and B; compare
curves 1 and 2) as demonstrated in the previous
section. p53 protein was added to the reaction mixture containing 2 nM Rh'-SO 5 min prior to basic peptide BD, and FCS
measurements were taken 2-5, 10-20, and 30-40 min thereafter.
Unexpectedly, GST-p53 added to the incubation mixture at 12 and 37 nM concentrations inhibited DNA aggregation induced by BD
(Fig. 6A, curves 3 and 4). No effect
of GST-p53 was registered at 3.7 nM concentration (Fig.
6B, curve 3). Practically identical results were
obtained at different time points after the initiation of the
reaction.

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Fig. 6.
Inhibition of DNA aggregation by p53: Effects
of GTP. Rh'-SO was incubated alone and in the presence or absence
of basic peptide BD, p53 proteins, and the nucleotide tri- and
diphosphates. The incubation mixture was analyzed by FCS. A,
normalized autocorrelation functions G( ) for: Rh'-SO (2 nM) alone (curve 1), Rh'-SO plus 3 µM BD (curve 2; D = 3878 ms),
Rh'-SO plus 12 nM GST-p53 + 3 µM BD
(curve 3), Rh'-SO plus 37 nM GST-p53 + BD
(curve 4), Rh'-SO plus BD + 37 nM GST-p53*
(curve 5), Rh'-SO plus BD + 37 nM GST-p53* + 5 mM GTP (curve 6), Rh'-SO plus 30 nM
GST-p53His273 + BD (curve 7), Rh'-SO plus 25 nM
GST-NTD + BD (curve 8), and Rh'-SO plus 25 nM
GST + BD (curve 9). p53 with or without GTP, as well as
other proteins were added to the reaction mixture 5 min before
(curves 3, 4, and 7-9) or 5-8 min
after* (curves 5 and 6) BD. B,
normalized autocorrelation functions G( ) for: Rh'-SO (2 nM) alone (curve 1), Rh'-SO plus 3 µM BD (curve 2; D = 3180 ms),
Rh'-SO plus 3.7 nM GST-p53 + BD (curve 3),
Rh'-SO plus 3.7 nM GST-p53 + 5 mM GTP + BD
(curve 4), Rh'-SO plus 3.7 nM GST-p53 + 5 mM GDP + BD (curve 5), Rh'-SO plus 3.7 nM GST-p53 + 5 mM GMP + BD (curve
6), Rh'-SO plus 3.7 nM GST-p53 + 5 mM
GMPPNP + BD + (curve 7), Rh'-SO plus 5 mM GTP
(curve 8), Rh'-SO plus 5 mM GDP (curve
9), Rh'-SO plus 5 mM GMP (curve 10), and
Rh'-SO plus 5 mM GMPPNP (curve 11). GST-p53 with
or without GTP, GDP, GMP, or GMPPNP was added to the reaction mixture 5 min before BD. Curves 1, 3, 4, and
6 and curves 2 and 7-9 in
A, as well as curves 1, 4, and
8-11 and curves 2, 3, and
5-7 in B are completely overlapping,
respectively. Concentrations of protein monomeric forms are given. Data
were recorded 10-20 min after the initiation of the reaction.
|
|
To determine whether p53 can reverse aggregation, GST-p53 (37 nM) was added to aggregated DNA 5-8 min later than BD in
incubation medium containing fluorescent oligonucleotide. In this
experiment p53 was found to partially dissociate preformed aggregates
(Fig. 6A, curve 5). Because dissociation of
aggregated DNA may require energy and the nucleotide triphosphates GTP
and ATP may regulate p53 interactions with DNA (16, 24), the effects of
GTP and its analogs on p53-mediated DNA disaggregation were tested. GTP substantially potentiated the ability of GST-p53 to disaggregate DNA
when GST-p53 (37 nM) was added to aggregate DNA 5-8 min
later than BD (Fig. 6A, compare curves 5 and
6). GST-p53 at 3.7 nM concentration did not
prevent DNA aggregation induced by BD in the absence of GTP but
strongly inhibited this reaction in the presence of GTP (Fig.
6B, curves 3 and 4); GST-p53 was added
to the reaction mixture 5 min prior to BD in this experiment. Besides
GST-p53, the wild type p53 protein, produced in bacteria, disaggregated
DNA and GTP potentiated this reaction (data not shown). GDP, GMP, and the nonhydrolyzable analog guanylyl-imidodiphosphate (GMPPNP) did not
potentiate the ability of 3.7 nM of GST-p53 to inhibit DNA
aggregation induced by BD (Fig. 6B, curves 5-7),
and these compounds and GTP themselves had no influence on the Rh'-SO
diffusion times (Fig. 6B, curves 8-11). GST-p53
(37 nM) also inhibited DNA aggregation by
poly-L-lysine (1.2 µM) in a GTP-potentiated
manner (data not shown).
GST-p53His273, p53 protein with a mutation in the core domain, did not
inhibit DNA aggregation induced by BD (Fig. 6A, curve 7). Preincubation of GST-p53 (12 nM) with PAb421
antibody (70 nM) for 30 min also reduced the ability of p53
to inhibit DNA aggregation induced by BD (data not shown). These
findings suggest that the core and CTD of the full-length p53 are
involved in DNA disaggregation. Because these domains in the form of
individual proteins aggregated DNA (Fig. 5D, curves
5 and 6), they probably act in a cooperative fashion in
full-length p53 when p53 prevents DNA aggregation. p53, activated by BD
or PAb421, bound a small fraction of radioactive BC oligonucleotide,
containing specific p53 DNA binding sites, in a band shift assay (Fig.
2). FCS was not, however, sufficiently sensitive to register this
fraction in the presence of an excess (>90%) of unbound fluorescent
oligonucleotide. The FCS data demonstrate that latent p53 prevents the
aggregation of most oligonucleotide molecules and releases them from
aggregates. This reaction requires that p53 contacts the majority of
oligonucleotide molecules in solution. Because no p53 complexes with
Rh'-SO (Fig. 5D, curve 2) and basic peptides
(Fig. 4, curve 2) were found in these experiments, p53-DNA
contacts were presumably short lasting.
Binding of Latent p53 to Long Double-stranded DNA via the Core and
CTD--
To generalize the FCS observation that the core and CTD of
latent p53 interact with DNA, interactions of these domains of full-length p53 with long DNA as a radiolabeled probe was tested in the
absence of basic peptides in a band shift assay. A ds 247-bp DNA
fragment with 15 copies of wild type p53 binding site, (PG; Fig.
7A, lanes 1-3 and
7-9), a ds 285-bp DNA fragment with 13 copies of mutant p53
binding site (MG; Fig. 7A, lanes 4-6), and another ds
686-bp DNA fragment (RD; Fig. 7B) were used as labeled probes. Incubation of the labeled DNAs with GST-p53 (Fig. 7,
A, lanes 1-6, and B, lane
1) or the wild type p53 protein, produced in bacteria (Fig.
7A, lanes 7-9), resulted in the formation of large complexes that did not enter polyacrylamide gels. Complex formation was dependent on protein concentration (Fig. 7A,
compare lane a with lane b and lane c
with lane d). Thus, latent p53 demonstrated no differences
in binding to long DNAs with wild type and mutant p53 binding sites,
respectively (Fig. 7A, lanes 1-6).

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Fig. 7.
Interaction of p53 with long ds DNA.
A, band shift analysis of the binding of GST-wild type p53
(lanes 1-6; lane a, 1.4 nM;
lane b, 5 nM) and wild type p53 (lanes
7-9; lane c, 5 nM; lane d, 10 nM) to the 32P-labeled ds 247-mer PG
(lanes 1-3 and 7-9; 0.3 nM) and
285-mer MG (lanes 4-6; 0.3 nM) DNA fragments
containing wild type and mutant consensus sites, respectively.
B, retardation of the labeled ds 686-mer RD fragment (0.05 nM) by the GST-wild type p53 (lanes 1 and
7-10, 1.4 nM), GST protein (lane
GST; 3.6 nM), GST-NTD (lane N; 2.5 nM), GST-p53His273 (lane m273; 1.4 nM), GST-CTD (lane C; 11 nM), and
GST-core domain (lane M; 2 nM). CMV-vector DNA
(lanes pl; lane a, 0.05 nM;
lane b, 0.25 nM) and ss 37mer (lane
n; 820 nM) N9 oligonucleotide (16) were used as
competitors. 32P-Labeled DNA fragments were incubated with
protein and analyzed on nondenaturing 5% polyacrylamide gels.
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|
To identify the region in p53 that interacts with long DNAs, the
binding of p53 domains to labeled ds 686-bp DNA fragment RD was
analyzed. Neither the GST-NTD nor the GST protein alone gave rise to
any complexes (Fig. 7B, lanes 2 and
3). The core domain produced stable complexes with the
labeled probe that did not enter the gel (lane 6). CTD also
bound long DNA; CTD-DNA complexes entered the gel or dissociated during
electrophoresis producing a smearing band (lane 5). The
GST-p53His273 mutant (lane 4) was approximately five times
less potent than GST-p53 (lane 1), demonstrating that the
core domain of the wild type p53 protein was involved in the
interaction with long DNA molecules; the residual binding likely
occurred because of CTD-DNA interactions. Plasmid DNA blocked the
binding to GST-p53 and the labeled DNA entered the gel (lanes 8 and 9). A short ss 37-mer oligonucleotide that blocks
CTD interactions with DNA (3, 4) was 104-fold less
efficient than plasmid DNA as a competitor (lane 10). These
observations indicate that the full-length p53 binds to long DNA
molecules via both the core domain and CTD.
 |
DISCUSSION |
Sequence- and Target-nonspecific Effects of Basic
Peptides--
p53 activation by peptides may be divided into two
processes. Peptides may, first, transform the latent form of p53 into
an "activated" form capable of binding to DNA target sequences and, second, stimulate binding of the activated form to DNA. The second process is illustrated by the observation that the p53 core domain, capable of binding to DNA, is activated by the basic CTD fragment, p53(361-383) (5). Previous studies indicated sequence specificity of
activation of latent p53 by the CTD fragments (4, 7). However, because
p53 is activated by two basic peptides BD and poly-L-lysine, which show no sequence similarity with these
fragments, there exists no sequence specificity (see also Ref. 25).
Besides poly-L-lysine, big dynorphin and its fragments
dynorphin A and B were used in the present study as model basic
peptides with apparently irregular alternations of basic residues. In
fact, BD and poly-L-lysine, which are larger and have
higher positive charge than p53(361-382), activated p53 more
efficiently. However, shorter peptides such as dynorphins A and B and
penta-L-lysine were unable to activate p53. The net
positive charge and overall peptide length seem to be the common
structural feature of activating peptides.
The activation of DNA binding by basic peptides is not unique for p53
and has been suggested to be a general in vitro phenomenon, which applies to BZLF1, C/EBP, and GAL4 (26) and YY1 (present study).
The mechanism of activation has not been identified, although it was
proposed that activating peptides themselves did not bind to DNA (26).
Electrostatic interactions of both DNA binding and transcription
activation domains with DNA contribute to DNA binding. For instance,
interactions of the negatively charged activation domains of the Fos
and Jun proteins with DNA result in bending of target DNA (27).
Multivalent cations such as Mg2+ and spermidine interfere
with these interactions and reduce DNA bending. p53 and YY1 regulate
gene transcription through a mechanism that may also involve DNA
bending (28-31). Peptide polycations may neutralize negative charges
on DNA, p53, and YY1 molecules and, thus, facilitate binding.
DNA Disaggregation by p53--
An important role of p53 in the
maintenance of integrity of the genome complementary to its functioning
as a transcription factor has been proposed (for a review see Ref. 1).
p53 in its latent state appears to be involved in DNA repair,
recombination, and replication via its ability to catalyze DNA
renaturation or strand exchange or via its exonuclease activity. DNA
molecules can undergo intermolecular aggregation into ordered, highly
condensed and thermodynamically stable states in solutions where
DNA-DNA interactions prevail, for instance in the presence of
polycations or upon confinement of DNA into a limited space, such as an
intracellular highly crowded environment (32). Liquid crystalline DNA
organization was observed in dinoflagellate chromosomes (33) and sperm
cells (34). Many proteins involved in DNA recombination can promote DNA
aggregation, and the formation of hybrid DNA intermediates may be
facilitated in DNA aggregates (35). p53-mediated inhibition of DNA
recombination (1) may be based on its ability to inhibit DNA
aggregation, a necessary event in DNA hybrid formation.
It has been shown that the nucleotide triphosphate GTP inhibits DNA
renaturation and strand exchange catalyzed by p53 (16) and that ATP
binds to p53 at its C terminus (36). Furthermore, the p53 DNA binding
function may be regulated by an ATP/ADP molecular switch, and p53 has
intrinsic ATPase and GTPase activities (24). We observed that only GTP,
but not GDP, GMP, and the nonhydrolyzable analog GMPPNP, potentiates
the ability of p53 to disaggregate DNA. This suggests that DNA
disaggregation induced by p53 is an energy-dependent process.
Binding of Latent p53 to Long but Not to Short DNA
Molecules--
p53 may bind preferentially to long (247-mer and
longer) but not to short (30-50-mer) DNA molecules due to a greater
number of nonspecific binding sites in long DNA, allowing the
accommodation of several p53 molecules. Another explanation is that p53
complexes have longer half-life with long DNA. The half-life may depend on the time p53 slides along a DNA molecule from its initial binding site to the DNA terminus where it dissociates from DNA. Long-living p53
complexes with long DNA have been observed in a band shift assay (Fig.
7), whereas short-lasting p53 interactions with short DNA are evident
by our observation that p53 dissociates and maintains in the
nonaggregated state practically all Rh'-SO molecules aggregated by BD
(Figs. 5 and 6).
Conversion of Latent p53 to the Form Competent for Specific DNA
Binding--
In the allosteric model of p53 latency, the interaction
of CTD with the core domain locks the p53 molecule in a state latent for binding to DNA (4). Direct evidence that CTD inhibits the core
domain binding to DNA is still missing. The steric model of p53 latency
is based on the fact that latent p53 does not bind to the consensus DNA
sequence in the presence of nonspecific DNA competitors but binds to
this sequence in their absence (9, 10). The latter experiments were
performed with p53, prepared from baculovirus-transfected insect cells
where p53 is partially activated (37). Our observation that p53,
produced in bacteria, is latent for binding to the consensus sequences
either in the presence or in the absence of nonspecific DNA does not
agree with the steric model of p53 latency.
Besides interaction with specific DNA sequences, p53 interacts via its
core with internal segments of long ss and ds DNA (8, 17), plasmid DNA
damaged by ionizing radiation (38), insertion/deletion lesions in DNA
(39), and supercoiled DNA (40). The crystal structure of p53 core,
bound to consensus and nonconsensus DNA sequences, has been determined
(41). The p53 core binds to these sequences with similar docking
arrangements, but the nonconsensus complex made fewer contacts with the
bases compared with the consensus complex. Thus, there was no contact
of Arg280 with guanidine of the invariant C-G pair of the
pentamer consensus [PuPuPuC(A/T)-(T/A)G], which is the most critical
contact in the consensus complex (29). Paradoxically, the full-length
p53 capable of binding to long ss and ds DNA via the core domain (8,
17) was latent for binding to the consensus sites (Ref. 7 and present study). These observations seem to be in conflict with the two described models because none of them allows the interaction of the
core of latent p53 with DNA.
In the absence of other satisfactory explanations of these
contradictions, we suggest that CTD, when it binds to DNA, prevents the
interaction of the core of the full-length p53 with specific, but not
with nonspecific DNA sequences (Fig. 8).
Two possible scenarios may be envisaged. First, CTDs, bound to DNA, may
create steric clashes between core domains in p53 tetramer-DNA
complexes. These clashes may prevent a proper orientation and
positioning of core domains that are necessary for a productive binding
to consensus sequences. Second, CTDs, directly linked with H2
-helices, may change the orientation of the helices by pulling them
toward the CTD-DNA contact sites. Resulting changes in positions of
Arg280 in H2
-helices critical for recognition may
prevent the binding to consensus sequences. Modifications of CTD by
phosphorylation or acetylation prevent CTD from interaction with DNA.
Thereafter, the rapprochement of core domains and their binding to the
adjacent DNA pentamers in p53 target sequences or the interaction of H2
-helices with these sequences may be allowed (Fig. 8). The binding of ss DNA ends or short ss DNA fragments to CTD may stabilize p53 in a
conformation, active for binding to target DNA sequences (Fig. 8).

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Fig. 8.
A scheme proposed for the activation of
sequence-specific DNA binding activity of p53. CTD basic sequence
and the core DNA binding sites are shown as black and
shaded segments. A, latent p53 binds similarly to
nontarget and target DNAs via both its core and CTD. CTD interactions
with DNA prevent the binding of the core domains to target DNA
sequences by either creating steric clashes between core domains in p53
tetramers or pulling H2 -helices directly linked with CTDs toward
the sites of CTD-DNA interaction. B, CTD modifications by
phosphorylation (PO4) and acetylation
(Ac) or (C) its binding to ss DNA ends block CTD
interaction with DNA. D, basic peptides aggregate DNA,
allowing interactions of the CTD with an adjacent DNA molecule.
Displacement of CTD from the core-DNA complex because of CTD
modifications (in B and C) or CTD binding to an
adjacent DNA molecule (in D) allow the rapprochement of core
domains and their binding to target DNA sites (consensus pentamers) or
the proper interactions of H2 -helices with target DNA sequences.
p53 dimers instead of tetramers are shown for simplicity.
|
|
CTD, bound to DNA via its basic segment, may make contact with the
adjacent surfaces of the core or N-terminal domains. CTD has been
suggested to interact with the proline-rich region, p53(80-94) (6,
42), and this interaction may potentially determine positions of
CTD-DNA contacts. Deletion of the proline-rich region leads to
activation of p53 sequence-specific DNA binding (42), possibly because
CTD of this p53 mutant protein is situated on DNA without creating
steric clashes with core domains or without inducing deformations of H2
-helices in p53 tetramer-DNA complexes.
A Putative Mechanism of p53 Activation by Basic Peptides--
The
basic peptide BD activated p53 binding to specific DNA sequences,
apparently without direct interaction with p53. This observation and
the correlation between DNA aggregation potentials of basic peptides
and their ability to activate latent p53 suggest that latent p53 is
converted to the form competent for sequence-specific DNA binding upon
interaction with DNA aggregates. In aggregates, DNA molecules are
packed tightly, and CTD and the core can interact with juxtaposed DNA
molecules (Fig. 8D). Such p53 binding would allow a proper
positioning of core domains and H2
-helices and consequently
formation of the core domain complexes with specific DNA sequences. It
would be important to test whether latent p53 binds via its core and
CTD to different DNA molecules or the same molecule in the presence of
basic peptides. In the absence of basic peptides, the interactions of
two p53 domains with the same DNA molecule would be more favorable
because of an electrostatic repulsion between negatively charged DNA
molecules. Studies of the crystal structure of full-length p53
complexes with DNA are required for verification of the steric,
allosteric, and the unifying two binding domain model of p53 latency.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Michael Schramm for critical
reading of the manuscript and valuable discussion, Drs. K. G. Wiman and G. Selivanova for p53 proteins and helpful discussion, Dr. K. Reznikov for discussion, Dr. L. Devi for pRC/CMV-Dyn plasmid, Dr. T. Shenk, for GST-C2 plasmid, and Dr. B. Vogelstein for PG-CAT and MG-CAT,
pC53-SN3, pC53-273, and pC53-248 plasmids.
 |
FOOTNOTES |
*
This work was supported by Swedish Cancer Society Grants
3935 and 5281 (to G. B. and T. J. E.), by Swedish
Medical Research Council Grants 12190 (to G. B.) and 3166 (to
L. T.), and by a fellowship from the Royal Swedish Academy of
Sciences (to I. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Permanent address: State Research Center of Virology and
Biotechnology Vector, Inst. of Molecular Biology, 633159 Koltsovo, Novosibirsk region, Russian Federation.

Present address: Harold L. Dorris Neurological Research
Center, Dept. of Neuropharmacology, Scripps Research Inst., 10550 North
Torrey Pines Rd., Mail SR-307, La Jolla, CA 92037.
§§
To whom correspondence should be addressed: Experimental Alcohol
and Drug Addiction Research Section, Dept. of Clinical Neuroscience, Karolinska Inst., S 171 76 Stockholm, Sweden. Tel.: 46-8-5177-5751; Fax: 46-8-5177-6180; E-mail: Georgy.Bakalkin@cmm.ki.se.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100482200
 |
ABBREVIATIONS |
The abbreviations used are:
CTD, C-terminal
domain;
FCS, fluorescence correlation spectroscopy;
BD, big dynorphin;
NTD, N-terminal domain;
Rh, rhodamine;
Rh-BD, tetramethylrhodamine-big
dynorphin;
ss, single-stranded;
ds, double-stranded;
ds-SO, double-stranded specific oligonucleotide;
Rh'-SO, double-stranded
5-carboxytetramethylrhodamine oligonucleotide with wild type p53
consensus binding sites;
Rh'-NO, double-stranded
5-carboxytetramethylrhodamine oligonucleotide mutant p53 consensus
binding sites;
GMPPNP, guanylyl-imidodiphosphate;
GST, glutathione S-transferase;
HPLC, high pressure liquid
chromatography;
bp, base pair(s).
 |
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