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INTRODUCTION |
Exposure of normal cells to agents that damage DNA initiates a p53
signal transduction cascade, resulting in either cell cycle arrest or
apoptosis (1). The interaction of p53 with DNA is thought to be
critical for its signaling because the majority of tumor-derived forms
of p53 have mutations in the central DNA binding domain (2), abrogating
the ability of p53 to bind its consensus DNA sites (3). To date, many
studies have focused on the sequence-specific interaction of p53 with
DNA, including those that have identified downstream transcriptional
targets and studies that describe post-translational modifications that activate p53 consensus site binding.
The ability of p53 to function as a transcriptional activator is
believed to be integral for its growth-suppressive properties (4, 5).
Sequence-specific transactivation is one of the most well understood
biochemical activities of p53. After cellular stress such as DNA damage
(6), hypoxia (7), viral infection (8), or activation of oncogenes such
as ras (9) and myc (10), p53 becomes
transcriptionally active. Once active, p53 induces, among many genes,
p21 (11), an inhibitor of cyclin-dependent kinases thought
to be necessary for the p53-dependent G1/S cell cycle arrest (12-15). The increase in p53-mediated transcriptional activity may be because of elevated levels of p53 in the cell (6, 16)
or increased sequence-specific binding ability (17, 18).
Post-translational modifications of p53, including phosphorylation by S
and G2/M phase cyclin-dependent kinase-cyclin
complexes (19, 20), DNA-dependent protein kinase (21),
protein kinase C (22), and casein kinase II (23), as well as C-terminal
acetylation (24) have been found to enhance sequence-specific DNA
binding in vitro. Furthermore, p53 has been shown to be
acetylated at its C terminus after exposing cells to ultraviolet or
ionizing radiation (18). This acetylation may be regulated by
phosphorylation of the p53 N terminus by either the ATM kinase or
DNA-dependent protein kinase (18, 21, 25, 26).
In addition to binding DNA containing consensus sites, p53 can interact
with nucleic acids in a sequence-independent manner. p53 can bind RNA
(27), short single-stranded DNA
(ssDNA)1 (28-30), and
double-stranded DNA containing nucleotide loops (31); these diverse
associations may be critical to p53 signal transduction. The ability of
p53 to bind ssDNA is of interest because this form of DNA is an
intermediate of both DNA damage and repair. Studies have correlated p53
signaling activation with both the timing and amount of DNA strand
breaks. Nelson and Kastan (32) have shown that p53 levels increased
after electroporation of enzymatically active restriction endonucleases
into cells. Microinjection of single-stranded circular phagemid or
circular DNA with a large gap into nuclei of normal human fibroblasts
induced a p53-dependent G1 arrest (33).
Jayaraman and Prives (34) reported stimulation of p53 consensus site
binding in vitro in the presence of short ssDNA fragments.
Further proof that p53 may directly interact with damaged DNA was
provided in a study reporting p53 binding to DNA fragments containing
insertion/deletion lesions (31). Also, confocal microscopy studies have
shown co-localization of p53 protein with sites of damaged DNA in
histological sections of human skin exposed to UV light (35).
Collectively, these reports suggest that p53 may be directly or
indirectly regulated by DNA damage intermediates. A direct interaction
of p53 with either DNA lesions or with proteins that bind damage
intermediates may be a relevant upstream event in the biochemical
engagement of the protein.
In this study, we analyzed the interaction of p53 with DNA fragments
containing insertion/deletion lesions. In contrast to many studies that
have shown p53 C-terminal binding to ssDNA or DNA lesions, we
demonstrate that an intact central domain and dimerization capability
are required for wild-type (wt) human p53 binding to IDLs. The results
of our binding analyses demonstrate that the affinity of p53 for DNA
fragments containing either an IDL or a consensus site is in the
pM range. Competition binding assays revealed that p53-IDL
complexes were dissociated with relatively low concentrations of
consensus site-containing DNA. However, the number and availability of
each DNA site immediately after DNA damage may promote p53 binding to
DNA lesions in lieu of sequence-specific DNA binding.
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EXPERIMENTAL PROCEDURES |
Expression of p53 Proteins--
Sf9 cells were infected
with either wt p53 or mutant p53273-expressing recombinant
baculovirus (kindly provided by C. Prives, Columbia University).
Protein extracts of infected cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet
P-40, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride (Sigma), 1 µM of E-64,
antipain (10 µg/ml), leupeptin (10 µg/ml), pepstatin A (10 µg/ml), chymostatin (10 µg/ml; Sigma), and
4(2-aminoethyl)benzenesulfonylfluoride (200 µg/ml;
Calbiochem-Novabiochem Corp.)) and sonicated, and extracts were
incubated on ice for 30 min. The protein lysates were centrifuged at
12,000 × g for 20 min at 4 °C. Supernatant was
collected and stored at
80 °C.
Extracts from Sf9 cells infected with baculoviruses expressing
various forms of histidine-tagged murine p53 (generously provided by P. Tegtmeyer, State University of New York) were prepared as outlined,
above except the lysis buffer used was DNA
Binding Buffer (DBB: 20 mM
Tris-HCl, pH 7.2, 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM dithiothreitol).
p53 expression vectors (4) were transformed into Saccharomyces
cerevisiae strain YPH681. Expression was induced by substituting galactose for dextrose in liquid cultures. Protein extracts were obtained by adding DBB and glass beads to the yeast pellets. Yeast were
lysed using a bead beater, and extracts were clarified by centrifugation at 12,000 × g for 10 min at
4 °C.
DNA Fragments--
The following oligonucleotides were used;
underlined sequences represent the triplet cytosines.
3C3,
5'-CGAACCCGTTCTCGGAGCACCCCTGCCCCAGCCCAACCGCTTTGGCCGCCGCCCAGCC-3'; C3,
5'-CGAACCCGTTCTCGGAGCACTGCCCCAGAACCGCTTTGGCCGCCGCCCAGCC-3'; negative control random sequence:
5'-CGAACCCGTTCTCGGAGCACTGCAGAACCGCTTTGGCCGCCGCCCAGCC-3'; consensus
binding site (nucleotides 2293-2332 of the p21 promoter (11), the
consensus site is underlined):
5'-TGGCCATCAGGAACATGTCCCAACATGTTGAGCTCTGGCA-3'. Oligonucleotides were purified on 10% PAGE/1× Tris borate-EDTA, 7 M urea gels, and end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (New England
BioLabs). Complimentary DNA strands were then annealed, and duplexes
were purified using 10% PAGE with 1× Tris acetate/borate-EDTA.
DNA Binding Assay--
To study the p53-IDL interaction, we used
an in vitro protein-DNA binding assay developed by McKay
(36) that allows for quantitative analysis. A monoclonal antibody that
recognizes the N terminus of p53, PAb1801, was chemically cross-linked
to protein A-Sepharose (PAS) with 52 mM dimethyl
pimelimidate (Pierce); this antibody does not interfere with the
oligomerization or DNA binding ability of p53. For assays using murine
p53 (N-terminally tagged with six histidine residues), a monoclonal
Penta-His antibody (Qiagen) was cross-linked to Protein G-Sepharose
(PGS). The antibody-PAS/PGS complex was added to yeast or baculoviral
protein extracts and mixed end-over-end for 1.5 h at 4 °C in
250 µl of DBB. Immunoprecipitated p53 was washed once with DBB,
followed by a 5 min end-over-end wash with 0.5 M NaCl in
Buffer B (5X Buffer B contains: 100 mM Tris-HCl pH 8.0, 5 mM EDTA, 50% glycerol), and finally by a 5 min
end-over-end wash in DBB. Subsequent analysis of these samples by
SDS-PAGE and silver staining showed immunopurification of p53 to
~95-98% homogeneity. The immunopurified protein was then mixed end-over-end for one h with 3.5 fmoles of
[32P]-end-labeled DNA fragments in 250 µl of DBB for
1 h at room temperature. After three washes with DBB, protein
components of the complex were digested with SDS/Proteinase K (VWR
Scientific) in TE8 (20 mM Tris pH 8.0, 10 mM
EDTA). DNA was phenol-chloroform extracted, ethanol precipitated, and
electrophoresed in 1× Tris-acetate/EDTA on a 10% PAGE at 100 V. Gels
were fixed in 5% methanol, 5% acetic acid before drying and exposure
to film. DNA was quantified using a 445 SI PhosphorImager (Molecular
Dynamics) and an Instant Imager (Packard Instruments). Alternative
processing of the protein-DNA complexes for protein detection involved
adding Laemmli sample buffer to the final complex and subjecting
samples to 10% SDS-PAGE electrophoresis. Gels were stained with
GelCode blue stain reagent (Pierce).
Saturation Binding Assays--
DNA binding assays were performed
as outlined above using 0.1 pmol of wt p53 protein immunopurified from
Sf9 cells and the indicated amounts of consensus or
C3 DNA fragment. For each DNA input, the nonspecific
binding component was determined by performing side-by-side assays with
the PAb1801-protein A-Sepharose complex incubated with protein from
mock-infected Sf9 cells. After final washes, the tubes were
counted in a scintillation counter.
Competition Binding Assays--
DNA binding assays were
performed as outlined above. The equilibrium DNA binding condition of
C3 was used (0.1 pmol of p53, 22 pM
C3 DNA). After counting the initial bound cpm, radiolabeled C3 fragment bound to p53 was competed by the addition of
the indicated amounts of unlabeled DNA in 250 µl of DBB. After 1 h of incubation at room temperature, complexes were washed three times
with DBB, and the tubes were counted in a scintillation counter.
Prism software (GraphPad) was used for analyses of binding data. The
percent incorporation of each radiolabeled DNA fragment, the
nonspecific binding component, and radioactive decay rate of the DNA
fragments were corrected for in the analyses.
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RESULTS |
To study p53-IDL interactions, we used an in vitro
protein-DNA binding assay developed by McKay (36) that allows for
quantitative analysis. The DNA lesions in this study were the same that
Lee et al. (31) used to demonstrate p53 binding to IDLs
through gel shift and electron microscope analyses. Random sequence,
double-stranded DNA fragments with either one (C3) or three
(3C3) sets of triplet cytosines in one of the strands were
used; the extra bases caused a nucleotide loop in the DNA duplex.
Full-Length p53 Binds to DNA Fragments Containing IDLs--
p53
protein produced in either baculoviral or yeast overexpression systems
bound to DNA fragments containing IDLs at a level proportional to the
number of triple cytosine loops in the DNA (compare middle
and right panels of Fig. 1).
Comparable p53 binding activity was also seen using DNA fragments of
different sequence context that contained a triple cytosine loop (data
not shown). p53 did not bind to a control DNA duplex without triplet
cytosines (see the first panel of Fig. 1). There was minimal
DNA binding activity detected with control immunoprecipitates from
yeast and Sf9 extracts lacking p53 protein (Fig. 1, see
Protein Con lanes). The p53273 tumor-derived
mutant form of p53 was also tested in the assay and did not exhibit
significant binding to DNA fragments containing IDLs.

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Fig. 1.
p53 binds DNA containing insertion/deletion
mismatches. Binding assays were performed with the DNA fragments
shown and p53 protein preparations from Sf9 or yeast cells.
Control DNA (Con DNA) represents a random sequence,
double-stranded DNA fragment. The DNA fragments containing IDLs were
formed by inserting either three extra cytosines (C3) or
three sets of three extra cytosines (3C3) into one of the
DNA strands. Protein control (Protein Con) represents an
immunoprecipitate of a crude protein lysate from Sf9 or yeast
cells not engineered to express p53 protein. p53273
represents the Arg His273 mutant. An equivalent amount
of p53 protein was used in each assay, and the results are
representative of five independent experiments.
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Tumor-derived Mutant Forms of p53 Lack C3 Binding
Activity--
The lack of IDL binding by the p53273
protein prompted us to screen other p53 proteins with point mutations
in the central domain. Four mutant p53 proteins representative of
tumor-derived forms were produced in yeast and immunopurified, and
equivalent amounts of protein were analyzed in the binding assay (Fig.
2B). We found that all four
mutant p53 proteins had <5% of the wt p53 binding activity to the
C3 IDL (Fig. 2A). These data suggest that
full-length p53 requires an intact central domain to bind DNA fragments
containing IDLs. However, these results do not rule out the possibility
that the p53 C terminus, as a separate entity, can also bind IDLs, as
previously reported by Lee et al. (31).

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Fig. 2.
Tumor-derived mutant p53 proteins lack
C3 binding activity. A, the results of binding
assays performed with the C3 DNA fragment and protein
immunopurified from yeast cells engineered to express the following p53
point mutants: Val143 Ala, Arg175 His,
Arg248 Trp, and Arg273 His.
Quantification of the bound radiolabeled DNA is presented in the
histogram. B, level and purity of the proteins used in the
assay as visualized on a Coomassie Blue-stained SDS-polyacrylamide gel.
Results are representative of five independent experiments.
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Deletion of the C-Terminal 40 Amino Acids of p53 Abrogates
C3 Binding--
To study the role of the p53 C terminus in
IDL binding, engineered proteins with successive deletions of the C
terminus were overexpressed in yeast, and equivalent amounts of
proteins were tested for C3 DNA fragment binding (Fig.
3B). Up to 40 amino acids could be deleted from the C terminus of p53 without loss of protein binding to IDLs (Fig. 3A; see 1-353). In fact,
deletion of 20 amino acids from the C terminus increased binding
activity by more than 2-fold over wt p53 (Fig. 3A; see
1-373). However, deletions of 50 or 60 amino acids resulted
in 90% loss of IDL binding (Fig. 3A; see 1-343
and 1-333).

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Fig. 3.
Deletion of the C-terminal 40 amino acids of
p53 abrogates C3 binding. A, the results of
binding assays performed with the C3 DNA fragment and
C-terminal-truncated p53 proteins immunopurified from yeast cells.
Quantification of the bound radiolabeled DNA is presented in the
histogram. B, level and purity of the proteins used in the
assay as visualized on a Coomassie Blue-stained SDS-polyacrylamide gel.
Results are representative of five independent experiments.
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The loss of IDL binding observed with deletions of 50 or more amino
acids from the C terminus suggested that either the intrinsic C-terminal sequence was required for IDL binding or the oligomerization domain, which is encompassed within residues 312-365 of human p53
protein (37), must be intact for this p53 activity. To determine which
of these two properties was necessary for p53-IDL binding, we used
chimeric proteins containing various C-terminal-truncated p53 fused to
the coil-coil (CC) dimerization domain of the yeast transcription
factor GCN4 (residues 249-281) (38) for further analyses (Fig.
4A). p53-CC fusion proteins
with deletions up to 100 amino acids from the p53 C terminus were able
to bind IDLs (Fig. 4B). In fact, the p53-CC fusion proteins
293CC, 323CC, 333CC, 343CC displayed binding activities ~2-4-fold
greater than wt protein. However, despite the enhanced binding seen
with these fusion proteins, a single point mutation in the central
domain of the p53 portion of the chimeric protein resulted in 90% loss
of IDL binding (Fig. 4A, compare 343CC to
347CC175). Fusion proteins with p53 C-terminal deletions
greater than 100 amino acids (273CC and 283CC) had less than 10% wt
binding activity; these p53 deletions disrupted the central DNA binding domain, which is encompassed by residues 100-300 (39). Thus, the data
suggest that full-length p53 binding to IDLs requires an intact central
domain and that the entire C terminus of p53 is functionally
replaceable with a foreign dimerization domain in these assays

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Fig. 4.
The dimerization domain of p53 can be
replaced by the coiled-coil domain of the yeast transcription factor
GCN4. A, schematic representing the construction of
C-terminal deletions of p53 fused to the CC dimerization domain of the
yeast transcription factor GCN4. I-V represent
evolutionarily conserved regions in p53. aa, amino acids.
B, the results of binding assays performed with the
C3 DNA fragment and protein immunopurified from yeast cells
engineered to express the indicated proteins. Quantification of the
bound radiolabeled DNA is presented in the histogram. C,
level and purity of the proteins used in the assay as visualized on a
Coomassie Blue-stained SDS-polyacrylamide gel. Results are
representative of five independent experiments.
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C3 Binding Activity of Full-length p53 Versus the
C-terminal Fragment--
Previous studies have reported that the C
terminus alone can bind IDLs (31). To determine the relative binding
activities of full-length p53 and the C-terminal fragment, we assayed
the C3 binding properties of wt human p53 and three forms
of murine p53 produced in Sf9 cells: full-length p53, amino
acids 1-360, and amino acids 315-390 (40). Binding assays were
performed with equimolar amounts of p53 protein incubated with the
C3 DNA fragment (Fig. 5).
Murine and human wt p53 had equal C3 binding activity.
Similar to our results with human p53 C-terminal truncation mutants
(Fig. 3A), partial deletion of the C terminus of murine p53
significantly lowered C3 binding ability. Consistent with the results of Lee et al. (31), we found that the C-terminal fragment of p53 could bind C3, albeit with less than 10%
full-length p53 IDL binding activity.

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Fig. 5.
C3 binding activity of
full-length p53 and the C-terminal fragment. The results of
binding assays performed with the C3 DNA fragment and
protein immunopurified from a crude extract of Sf9 cells
engineered to express the indicated proteins. Quantification of the
bound radiolabeled DNA is presented in the histogram. Results are
representative of three independent experiments. Protein
Con, protein control.
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Saturation Binding of p53 to DNA Fragments Containing
Consensus Sites or IDLs--
The significance of p53 binding to its
consensus DNA site is supported by many biological studies (41) and the
report of nM affinities for p53 binding to derivatives of
its consensus site (42). The lack of significant C3 binding
by tumor-derived mutant forms of p53 suggests this activity, like the
p53 consensus binding, may be biologically relevant. To determine
whether IDL binding occurs in a physiologic range, we performed
saturation binding assays to compare the affinity of full-length, wt
human p53 for its consensus site with that for C3. The p53
binding site in the p21 promoter (nucleotides 2293-2332) was used for
consensus binding analyses. Using 0.1 pmol of p53 protein and
increasing amounts (0.5 to 750 pM) of labeled DNA, we
determined that the KD for p53 binding to its
consensus site was 31 pM (Fig. 6A), and the
KD for p53 binding to the C3 DNA was 45 pM (Fig. 6B). Transformation of the saturation
binding isotherms into linear Scatchard plots facilitates the
comparison of p53 binding to the two DNA fragments (Fig.
6C). Although the binding affinity and capacity of p53 for
its consensus site are higher than those for C3, both
KD values represent physiologically significant
binding.

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Fig. 6.
p53 affinity for DNA fragments containing
IDLs. Binding assays were performed by incubating 0.1 pmol of p53
with increasing amounts of either A, p53 consensus site
representing nucleotides 2293-2332 of the p21 promoter; or
B, C3 DNA fragment. KD
represents the concentration of p53 required to reach half-maximal
binding of the DNA. Bmax is the maximal binding.
The equation used to plot the data represents a rectangular hyperbolic
function, indicative of binding that follows the mass action law:
Y = Bmax × X/(KD + X). Scatchard analysis
was perfomed on the saturation-binding isotherms, and the linear
representation of the data is seen in panel C. Results are
representative of three independent experiments.
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Competition of p53 DNA Binding--
To confirm the relative
affinities determined using saturation binding curves and to study the
DNA binding property of p53 when both a consensus DNA site and IDL DNA
site were available, competition binding assays were performed. Under
equilibrium conditions, p53 was first bound to the radiolabeled
C3 DNA fragment, and increasing concentrations of either
unlabeled consensus site, C3, or a random sequence dsDNA
fragment were added. p53 was efficiently competed off of C3
after addition of consensus site DNA with an EC50 of 0.14 nM (Fig. 7). The
EC50 for the C3 DNA fragment competition of prebound C3 DNA was 0.50 nM (Fig.
7). In contrast, a random sequence dsDNA fragment was an ineffective
competitor for C3 binding. The EC50
for this latter competition assay could not be accurately assessed
because 50% competition was difficult to attain (Fig. 7). Also, in
these assays, ssDNA was not an effective competitor as compared with
C3 (data not shown). These data demonstrate that p53 has a
higher affinity for its consensus site as compared with an IDL site and
will likely bind to the former if both DNA sites are present, with all
other variables remaining constant.

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Fig. 7.
Dissociation of p53-IDL complexes by
competitor DNA. Competition assays were performed at equilibrium
binding conditions of p53 binding to the C3 fragment. After
binding the 32P-end-labeled C3 DNA fragment,
increasing concentrations of either unlabeled control, consensus site,
or C3 DNA fragments were incubated with the complex.
EC50 represents the concentration of competitor that
reduced p53-DNA binding by 50%. Competition assays were analyzed using
GraphPad Prism software. The equation used to plot the data is
indicative of competition of binding to a single site:
Y = Bottom + (Top-Bottom)/(1 + 10(X EC50)).
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DISCUSSION |
In this study, we analyzed p53 binding to DNA fragments containing
IDLs. We found that full-length p53 requires an intact central domain
and dimerization capability to bind IDLs. Tumor-derived mutant forms of
p53 lost IDL binding, and C-terminal truncation mutants still bound
IDLs if the protein maintained dimerization capability. The
pM dissociation constant that we observed for p53 binding
to IDLs provides evidence for potential in vivo relevance.
Previously, the C terminus of p53 (amino acids 311-393) was shown to
bind IDLs (35). Reed et al. (43) also find that the C
terminus of p53 binds nonspecifically to dsDNA (43). These data
contribute to a model suggesting that the p53 C terminus negatively
regulates the sequence-specific binding activity of the protein. When
the p53 C-terminal end is deleted, phosphorylated, acetylated, or bound
to antibody, peptide, or single-stranded DNA, p53 binding to its
consensus site is stimulated (18, 24, 34, 44). Although we demonstrated
that the C terminus of murine p53 could bind IDLs, an equimolar amount
of murine p53 lacking the C terminus had greater affinity for IDLs than
the C-terminal fragment alone.
Our finding that the C terminus of p53 is not required for IDL binding
is in agreement with the study of Parks et al. (45) demonstrating that p53 binding to IDLs was unaffected by PAb421, a
monoclonal antibody that binds the C-terminal amino acids 371-380. Also, Bakalkin et al. (28) suggest that the domain of p53
responsible for nonspecific DNA binding depends on the DNA substrate;
the C terminus of p53 binds single-stranded ends of DNA, whereas the central DNA binding domain of p53 binds internal ssDNA segments (28).
The identity of the p53 binding domain(s) for various forms of DNA
damage may suggest distinct roles for p53 in DNA damage-signaling
pathways or DNA repair.
Several reports suggest that p53 is an important determinant in
nucleotide excision repair (NER). Using cells derived from patients
with Li-Fraumeni syndrome, Ford and Hanawalt (46) show that the
efficiency of global NER was dependent on p53 status. Compared with
cells heterozygous for p53, homozygous mutant p53 cells exhibited
global NER deficiency; however, transcription-coupled repair was
unaffected by the p53 status. The ability of p53 to bind RPA (47) and
subunits of TFIIH (48), both of which are essential components of NER,
suggests that p53 may play a direct role in NER. These protein
associations are among those that define similarities between p53 and
XPA, the damage recognition and binding component of NER (49). XPA
binds DNA cooperatively with RPA (50) and once complexed with DNA,
recruits TFIIH to the site of damage (51). p53 binding to IDLs may also
be stimulated by RPA. XPA can bind to various damage lesions (49), and
we have observed p53 binding to DNA fragments containing a cholesterol adduct (data not shown), an artificial DNA lesion used in in
vitro DNA repair assays (52).
Although we show that p53 has higher affinity for its consensus site as
compared with a DNA lesion, the temporal availability of p53 consensus
binding sites must be considered. Wu and Levine (53) have reported that
the p21 gene is the first measurable target of p53 transactivation
after high dose UV irradiation. However, p53-mediated induction of p21
gene expression does not occur until 2 to 5 h post-irradiation. In
agreement with this result, using in vivo footprinting, Chin
et al. (54) did not observe significant DNaseI cleavage
protection of the p21 promoter until 2 h after exposure of cells
to 20 gray of ionizing radiation. After exposure of cells to 1 gray of
ionizing radiation, 2 to 8 double-strand breaks/genome have been shown
to occur (55). In separate studies, Ji et al. reported that
treatment of cells with malondialdehyde (an endogenous product of lipid
peroxidation) resulted in the formation of 300 M1G-DNA
adducts/genome and subsequent elevation of p53 activity several hours
later (56). Thus, immediately after exposure of cells to genotoxic
agents, the number of DNA lesions in a cell would likely exceed
accessible p53 consensus sites, and p53 binding to DNA lesions may
occur. The pM dissociation constant that we report for p53
binding to IDLs is consistent with the physiological concentrations of
both p53 and DNA lesions in the cell after DNA damage. However, once
DNA repair is initiated, the availability of consensus sites would
likely increase, and the higher affinity of p53 for these sites would
shift the binding equilibrium.
Our understanding of the biochemical activities that are required for
p53 tumor-suppressive activities has increased enormously in recent
years. However, the role of DNA damage intermediates and the network of
signaling pathways by which cells activate p53 in the overall response
to DNA damage is not well defined. Determining if p53 is directly
activated by DNA damage intermediates and understanding how the
sequence-specific and sequence-nonspecific DNA binding activities of
p53 are integrated will contribute to our knowledge of how human cells
respond to a wide range of DNA lesions.