DNA modification with cisplatin affects sequence-specific
DNA binding of p53 and p73 proteins in a target
site-dependent manner
Hana Pivon
ˇ
kova
´
1
, Petr Pec
ˇ
inka
1
, Pavla C
ˇ
es
ˇ
kova
´
2
and Miroslav Fojta
1
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
2 Masaryk Memorial Cancer Institute, Brno, Czech Republic
The tumor suppressor protein p53 is known as a tran-
scription factor involved in cell cycle control [1–3]. It
plays a crucial role in preventing malignant transfor-
mation of a cell via induction of cell cycle arrest or
programmed cell death in response to stress conditions
(e.g. DNA damage). The functions of p53 are closely
related to sequence-specific recognition of response ele-
ments [p53 DNA-binding sites (p53DBSs)] in promot-

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(Received 25 April 2006, revised 18 July
2006, accepted 17 August 2006)
doi:10.1111/j.1742-4658.2006.05472.x
Proteins p53 and p73 act as transcription factors in cell cycle control, regu-
lation of cell development and ⁄ or in apoptotic pathways. Both proteins
bind to response elements (p53 DNA-binding sites), typically consisting of
two copies of a motif RRRCWWGYYY. It has been demonstrated previ-
ously that DNA modification with the antitumor drug cisplatin inhibits
p53 binding to a synthetic p53 DNA-binding site. Here we demonstrate
that the effects of global DNA modification with cisplatin on binding of
the p53 or p73 proteins to various p53 DNA-binding sites differed signifi-
cantly, depending on the nucleotide sequence of the given target site. The
relative sensitivities of protein–DNA binding to cisplatin DNA treatment
correlated with the occurrence of sequence motifs forming stable bifunc-
tional adducts with the drug (namely, GG and AG doublets) within the
target sites. Binding of both proteins to mutated p53 DNA-binding sites
from which these motifs had been eliminated was only negligibly affected
by cisplatin treatment, suggesting that formation of the cisplatin adducts
within the target sites was primarily responsible for inhibition of the p53 or
p73 sequence-specific DNA binding. Distinct effects of cisplatin DNA
modification on the recognition of different response elements by the p53
family proteins may have impacts on regulation pathways in cisplatin-
treated cells.
Abbreviations
cisPt-DNA, cisplatin-modified DNA; CTDBS, C-terminal DNA-binding site; EMSA, electrophoretic mobility shift assay; fl, full length;
IAC, intrastrand crosslink; oligo, oligonucleotide; p53DBS, p53 DNA-binding site.
FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4693

isoforms of the p73 protein have been found that differ
in the structure of their N-terminal and ⁄ or C-terminal
domains [21,22]. Although it was originally supposed
that the p53 homologs have redundant functions in the
regulation of gene expression, more recent data suggest
that p73 and p63 proteins do not act as ‘classic’ tumor
suppressors, but rather play important roles in the
regulation of cell development and differentiation
[21,23]. Nevertheless, some observations suggest that
p73 is involved in the cellular response to DNA dam-
age and in apoptosis control [24,25].
Cisplatin [cis-diamminedichloroplatinum(II)] is a
clinically used anticancer agent [26,27]. The drug binds
covalently to DNA, forming several kinds of adduct,
among which the most abundant are intrastrand cros-
slinks (IACs) between neighboring purine residues.
The spectrum of cisplatin adducts identified in globally
modified chromosomal DNA comprises about 50%
of 1,2-GG IACs, 25% of 1,2-AG IACs, 10% of
1,3-GNG IACs and interstrand crosslinks, and another
2–3% of monofunctional adducts. It has been found
that cisplatin cytotoxicity is related mainly to the IACs
that induce significant changes in the DNA
conformation, including bending and unwinding of the
DNA double helix [26,28]. The lesions are selectively
bound by a variety of nuclear proteins, and it was pro-
posed that these interactions are important for the
anticancer activity of the drug [26,29,30].
Interactions of the p53 protein with cisplatin-modi-
fied DNA (cisPt-DNA) have recently been studied [31–

motifs was not significantly affected by the DNA cis-
platination. Formation of the cisplatin adducts outside
the p53DBSs did not apparently influence p53
sequence-specific DNA binding.
Results
To analyze the sequence-specific DNA binding of p53
and p73 proteins, we designed 50-mer oligonucleotide
substrates bearing various p53DBSs (Fig. 1). In most
experiments, we used a C-terminally truncated, consti-
tutively active p53(1–363) to eliminate the sequence-
nonspecific p53 interactions with the cis-platinated
DNA, which have been shown to be mediated pri-
marily by the p53 C-terminal DNA-binding site
(CTDBS) [34]. In the presence of competitor nonspe-
cific DNA, sequence-specific binding of the p53(1–
363) protein to the
32
P-labeled 50-mer targets resulted
in the appearance of a distinct retarded band R
53
in
the polyacrylamide gel (Fig. 2). Binding of the p73b
Cisplatin effects on p53 ⁄ p73 DNA recognition H. Pivon
ˇ
kova
´
et al.
4694 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS
Fig. 1. Scheme of DNA substrates used in this work. All p53 DNA-binding sites (p53DBSs) were placed in the center of 50-mer oligonucleo-
tides (oligos), being flanked with the sequences shown on the top (the same stretches flank the p53DBSs in the pPGM1 and pPGM4 plas-

H. Pivon
ˇ
kova
´
et al. Cisplatin effects on p53 ⁄ p73 DNA recognition
FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4695
and p73d proteins to the DNA targets caused the for-
mation of analogous retarded bands (denoted as R
73b
or R
73d
, respectively; lanes 3 and 4 in Fig. 2A) whose
mobilities reflected different molecular weights of the
p73 isoforms. To verify the specificity of the band
shifts for DNA complexes with the proteins studied,
we used the band supershift assay with antibodies
against the p53 or p73 proteins. Addition of the DO-
1 antibody [17,38,39] mapping to the N-terminus of
the p53 protein resulted in further retardation of the
specific p53–DNA complexes (lane 5 in Fig. 2A),
producing two supershifted bands (SR
53
; Fig. 2A).
Formation of the two bands corresponded to two
possible stoichiometries of the antibody–p53 complex,
involving either one or two antibody molecules bound
per p53 tetramer [16,39]. For supershifting of DNA
complexes with the p73 constructs, which were tagged
with hemagglutinin (HA), we used antibody to HA
and obtained analogous band patterns to those

fered significantly. The steepest decrease in p53–DNA
binding with degree of DNA modification was exhib-
ited by the mdm2 target. The R
53
band due to the
p53–mdm2 complex exhibited only 10% intensity for
r
b
¼ 0.02, compared to the R
53
band due to protein
binding to the same but unmodified substrate (the r
b
value refers to the number of platinum atoms per total
DNA nucleotide). In contrast, the PGM1 and p21 tar-
gets retained 75% and 53% of the p53-binding capa-
city at r
b
¼ 0.02, respectively (Fig. 3A). Increasing the
DNA modification degree to r
b
¼ 0.04 resulted in a
decrease of p53–p21 binding to 42%, whereas the
PGM1 site bound only 16% of the protein, compared
to the same but unmodified p53DBS. At r
b
¼ 0.06, all
mdm2, PGM1 and p21 targets exhibited very weak
p53 binding (about 4% for mdm2 and PGM1 and
10% for p21).

formation of the bifunctional cisplatin adducts were
removed from the p53DBS. In addition, we derived
another ‘unreactive’ p53DBS from the PGM1 target
(PGM4; Fig. 1) by replacing all guanine residues,
except for those at the strictly conserved positions
[4,5], by adenines. All of these mutated p53DBSs
(when cisplatin-unmodified) exhibited sequence-specific
p53 binding comparable to that of the parent targets
(Fig. 2B).
Cisplatin effects on p53 ⁄ p73 DNA recognition H. Pivon
ˇ
kova
´
et al.
4696 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS
We studied how the cisplatin treatment influences
interaction of the p53(1–363) protein with the
mutated target sites. The 50-mer oligos containing
sequences p21a, p21b or PGM4 were treated with
cisplatin as above. DNA modification to r
b
¼ 0.02
resulted in a decrease of p53 binding to the p21a tar-
get by about 15%, which represented weaker inhibi-
tion than observed with the p21 target (25% decrease;
Fig. 3). More conspicuous differences between the
p21a and p21 targets appeared at r
b
¼ 0.04 (35% or
58% inhibition, respectively). At r

IACs within the p53DBSs due to the occurrence of
the GG, AG and ⁄ or GNG motifs, the stronger the
inhibition of the p53 sequence-specific DNA binding
to these targets caused by the DNA treatment with
cisplatin.
AB
Fig. 3. Effects of DNA modification with cisplatin on p53(1–363) binding to various target sites: (A), natural p53 DNA-binding sites (p53DBSs)
mdm2 and p21, and the synthetic PGM1 sequence; (B) mutated p53DBSs PGM4, p21a and p21b (Fig. 1). The top panels show sections of
autoradiograms showing the R
53
bands corresponding to complexes of p53 with the 50-mer target oligonucleotides (oligos) (Fig. 2). The
extents of DNA modification with cisplatin (r
b
) are indicated. Other details are as in Fig. 2. The graphs show the dependence of relative p53
binding to the targets on the degree of DNA modification (data obtained from densitometric tracing of the autoradiograms; for each target
site, the intensity of the R
53
band resulting from p53 binding to unmodified DNA was taken as 1.0, and the intensities of bands correspond-
ing to p53 binding to the same but cisplatin-treated substrate were normalized to this).
H. Pivon
ˇ
kova
´
et al. Cisplatin effects on p53 ⁄ p73 DNA recognition
FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4697
We also performed parallel experiments with fl p53
(expressed in insect cells). Like p53(1–363), the fl pro-
tein was able to recognize sequence specifically all of
the targets tested (not shown). The effects of the
degree of DNA cis-platination on recognition of the

PGM1 and PGM4 target sites (not shown).
Competition experiments
We studied the influence of cisplatin DNA modifica-
tion on the competition between two p53 target sites
for the protein (Fig. 6). The 474 base pair fragments
of plasmids pPGM1 or pPGM4 were used as the
sequence-specific competitors, and changes in p53(1–
363) binding to the
32
P-labeled 50-mer targets were fol-
lowed. We first tested the effect of the presence of the
competitor fragments (unmodified or treated with cisp-
latin) on p53 binding to the unmodified PGM1 probe
(Fig. 6A). Addition of either of the unmodified frag-
ments (70 ng per sample) resulted in a partial decrease
(by 35–45%) of the R
53
band intensities due to binding
of a portion of the p53 molecule to the competitor
p53DBS. Modification of the pPGM1 fragment with
cisplatin caused a reduction of its competitiveness,
which was manifested by increasing relative intensity
of the R
53
band yielded by the p53 complex with the
radiolabeled PGM1 probe. When the pPGM1 frag-
ment was cis-platinated to r
b
¼ 0.04 or 0.06, its pres-
ence had practically no effect on the R

¼ 0.02
can be attributed to partial loss of the competitiveness
of the pPGM1 fragment due to its modification, which
compensated for inhibition of p53 binding to the (rel-
atively less reactive) p21 target. When the mixture of
the p21b target with the pPGM1 competitor fragment
was treated with cisplatin in the same way, the inten-
sity of the R
53
band on the autoradiogram increased
with the degree of DNA modification. The increase
was already significant at r
b
¼ 0.02. At r
b
¼ 0.04 or
0.06, the relative intensity of the R
53
band reached
about 90% of the value observed with unmodified
DNA (Fig. 6B). Such behavior reflected inhibition of
p53 binding to the competitor pPGM1 fragment due
to its cis-platination, whereas interaction of the protein
with the p21b target remained practically unaffected
Fig. 4. Effects of DNA modification with cisplatin on full-length p53
binding to the mdm2, PGM1 and PGM4 targets. For more details,
see Figs 2 and 3.
Cisplatin effects on p53 ⁄ p73 DNA recognition H. Pivon
ˇ
kova

untwisting as well as perturbation of hydrogen bond-
ing within the base pairs [26–28]. Cisplatin adducts
occurring within p53DBS can therefore be expected to
cause severe deformations of the binding site with con-
comitant destabilization of the p53–DNA interaction
(or even prevention of target recognition by the pro-
tein).
DNA binding of the C-terminally truncated
p53(1–363) protein
In this work, we studied the effects of cisplatin treat-
ment of various p53DBSs on the sequence-specific
binding of a truncated tetrameric p53 construct lacking
the C-terminal DNA-binding site, p53(1–363) [18,34].
This variant of the protein is known to be constitu-
tively active for sequence-specific DNA binding [16].
Models of p53 latency considering the (post-transla-
tionally unmodified) p53 C-terminus solely as a negat-
ive regulator of sequence-specific DNA binding [40,41]
have recently been questioned [42–44]. Instead, the p53
CTDBS has been proposed to cooperate with the core
domain in complex p53–DNA interactions. The
CTDBS has been shown to be essential for p53 bind-
ing to target sites adopting non-B conformations (such
as stem–loop or cruciform structures) [11,12,45–47].
On the other hand, p53 constructs lacking the CTDBS
are capable of efficient binding to short linear model
DNA targets in which the p53DBS is present in its
double-helical B-form. Moreover, deletion of the
CTDBS (amino acids 363–382) makes it possible to
separate sequence-specific p53 DNA binding from

the drug in the presence of an excess of nonspecific
competitor calf thymus DNA mimicking random-
sequence natural genetic material that can accommo-
date the cisplatin adducts regardless of the reactivity
of the particular p53DBS. The frequency of DNA
modification within the p53DBSs could thus be expec-
ted to reflect the known distribution of cisplatin
adducts in globally modified chromosomal (genomic)
DNA [26]. Provided that the cisplatin inhibitory effect
on p53 sequence-specific DNA binding is linked pri-
marily to the IACs formed within the target sites, the
susceptibility of different targets to the drug treatment
should correlate markedly with the incidence of the
cisplatin-reactive motifs in the p53DBSs. Such a corre-
lation was indeed found: the sensitivity of the target
sites to treatment with the drug followed the trend
mdm2 > PGM1 > p21 > p21a > p21b  PGM4, in
accordance with the number and kind of motifs suit-
able for formation of the IACs inside the p53DBSs
(Fig. 1).
AB
Fig. 6. Competition between two different
p53 target sites in globally cisplatin-modified
DNA for the p53(1–363) protein. In (A),
32
P-labeled, unmodified PGM1 50-mer was
mixed with cisplatin-treated competitor frag-
ments of plasmids pPGM1 or pPGM4 (and
with unmodified calf thymus DNA) prior to
addition of the p53 protein. When the com-

the intensities of the R
53
bands observed
for the unmodified targets in the absence of
the competitor fragments (first samples of
each set) were taken as 1. For other details,
see Figs 2 and 3.
Cisplatin effects on p53 ⁄ p73 DNA recognition H. Pivon
ˇ
kova
´
et al.
4700 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS
In the p21 50-mer target and its derivatives p21a
and p21b, the 5¢-neighboring guanines in the ‘top’
strand form another GG doublet with the first guanine
of the p53DBS (Fig. 1). Interestingly, the presence of
this reactive motif had no conspicuous effect on p53–
p21b binding in the cisplatin-treated DNA, as there
were no significant differences between the behavior of
p21b and that of PGM4 (lacking this boundary GG
doublet; Fig. 1). The results presented in this article do
not make it possible to decide whether a single cisplat-
in IAC, wherever it is within the p53DBS, can fully
abrogate p53 sequence-specific DNA binding, or whe-
ther the protein can recognize such a cis-platinated
site, albeit with lower affinity. Nevertheless, our data
show clearly that a single reactive motif located within
the 20 base pair recognition element (e.g. in p21;
Fig. 1) caused significant sensitivity of p53–p53DBS

mdm2 site towards cis-platination was connected with
the abundance of the highly reactive GG motifs rather
than with the location of the AG doublet within the
CWWG tetranucleotide. On the other hand, our pre-
liminary results (M. Fojta et al., unpublished data)
suggest that the behavior of cisplatin-treated target
sites possessing a single GG motif at various positions
may differ significantly (more details will be published
elsewhere).
Altered sequence-nonspecific interactions of the p53
protein with DNA due to its cis-platination outside the
p53DBSs might, in principle, influence recognition of
the target sites by the protein. Nevertheless, control
tests of binding of the p53(1–363) protein to unmodi-
fied PGM1, PGM4, p21 and p21b targets in the pres-
ence of unmodified or cis-platinated (r
b
¼ 0.06) calf
thymus competitor DNA revealed no apparent effect
of the competitor modification. This observation was
in agreement with the recently reported lack of ability
of p53(1–363) to recognize the nonspecific cisPt-DNA
[34]. Furthermore, we were interested in whether the
presence of cisplatin adducts within DNA stretches
flanking the p53DBSs affects the ability of p53 to bind
the specific sequence. The flanking segments in all 50-
mer substrates used in this work (Fig. 1) contain three
motifs expected to form the 1,2-IACs (one GG and
two AGs). Another two sets of 50-mer substrates, in
which the PGM1 or PGM4 sites were flanked by seg-

ˇ
kova
´
et al. Cisplatin effects on p53 ⁄ p73 DNA recognition
FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4701
DNA-binding site (besides the core domain) analogous
to the p53 CTDBS been identified. Our results showed
that both p73d and p73b bound efficiently to all
(unmodified) p53DBSs used in this work, and that
cisplatin treatment of p21, p21b (Fig. 5), PGM1 and
PGM4 (not shown) affected the p73 sequence-specific
DNA binding basically in the same manner as
observed with p53.
Possible impacts on gene expression
in cisplatin-treated cells
It has been well established that modification of DNA
with cisplatin affects fundamental processes such as
DNA synthesis and transcription [26]. The bifunctional
cisplatin DNA adducts slow down or block DNA or
RNA polymerization and can hamper the initiation of
DNA transcription [49]. Strong differential inhibition
of marker gene expression was observed in cells treated
with cisplatin [50]. Interestingly, expression of genes
with stronger promoters was strongly inhibited,
whereas some genes possessing weaker promoters were
induced. It was proposed that the strong promoters
were associated with accessible chromatin and there-
fore more easily modified by the drug [50]. However,
to our knowledge, no systematic study of the sensitiv-
ity of various promoters (and particularly those con-

latin seems to be rather complex, and its relationship
to the status of the p53 family proteins does not
appear to be straightforward.
The results of our in vitro binding experiments sug-
gest that the expression of various p53 downstream
genes might be differentially affected in the cisplatin-
treated cells, due to different susceptibilities of the
p53 response elements to modification with the drug.
The natural p53DBSs [6,7] differ significantly in this
respect. Among the 20 response elements recently
characterized by Weinberg et al. [7], GADD45 (a gene
taking part in DNA repair ) no GG, three AG
doublets) and the p21 5¢-site (a single GGG triplet)
Fig. 7. The effect of DNA modification with
cisplatin on p53(1–363) binding to p53 DNA-
binding sites (p53DBSs) flanked by stret-
ches either totally lacking sites reactive to
cisplatin (PGM1-AT, PGM4-AT) or involving
multiple reactive motifs (PGM1-GC,
PGM4-GC). The flanking stretches are
shown at the top; for the p53DBS,s see
Fig. 1. The experimental conditions are as in
Figs 2 and 3.
Cisplatin effects on p53 ⁄ p73 DNA recognition H. Pivon
ˇ
kova
´
et al.
4702 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS
appear to be the most ‘cisplatin-resistant’, whereas

Inhibition of sequence-specific p53 (or p73) protein
DNA binding due to formation of the cisplatin
adducts within its response elements could thus repre-
sent an important, but not the only, factor affecting
cellular regulation pathways in cells exposed to the
drug.
Experimental procedures
DNA samples
Synthetic 50-mer oligonucleotides containing different
p53DBSs (Fig. 1) were supplied by VBC Genomics
(Vienna, Austria). Plasmids pPGM1 and pPGM4 [deriva-
tives of pBSK(+) vector containing the PGM1 and PGM4
sites; Fig. 1] were prepared as previously described [17,33].
The 474 bp fragments of pPGM1 and pPGM4 (delimited
by PvuII restriction sites) were prepared using PCR and
purified with the Qiagen PCR Purification kit (Qiagen, Hil-
den, Germany). Restriction endonucleases were supplied by
Takara (Otsu, Japan), thermostable Pfu DNA polymerase
by Promega (Madison, WI, USA), PCR primers by VBC
Genomics and nucleotide triphosphates by Sigma (St Louis,
MO, USA).
DNA modification with cisplatin
DNA samples were incubated with cisplatin (Sigma) in
10 mm NaClO
4
at 37 °C for 48 h in the dark. Radioactively
(
32
P) labeled 50-mer substrates (10 lgÆmL
)1

Quick Coupled Transcrip-
tion ⁄ Translation System (Promega). Plasmids pcDNA3-
HA-p73b or pcDNA3-HA-p73d (1 lg) coding the respective
p73 isoforms (both HA-tagged at their N-termini) were
mixed with 1 lLof1mm methionine, 40 lL of TNT
Ò
T7
Quick Master Mix and nuclease-free water to a final vol-
ume of 50 lL. Samples were incubated at 30 °C for 90 min.
Protein concentrations were determined densitometrically
from Coomassie blue G-250-stained polyacrylamide gels.
DNA-binding assays
In all experiments, the p53 or p73 proteins were mixed with
the DNA substrates in 2 mm dithiothreitol, 50 mm KCl,
5mm Tris (pH 7.6) and 0.01% Triton X-100 (total volume
20 lL) and incubated on ice for 30 min. The protein ⁄ DNA
target site molar ratio (i.e. protein tetramers per radiolabe-
led 50-mer probe) was 5 ⁄ 1. The reaction mixture contained
50 ng of the
32
P-labeled oligo and 2 lg of nonspecific com-
petitor calf thymus DNA. After the incubation period, the
protein–DNA complexes were analyzed by electrophoretic
mobility shift assay (EMSA) in 5% native polyacrylamide
gel containing 30 mm Tris, 30 mm H
3
BO
3
, 0.7 mm EDTA
buffer (pH 8.0) at 4 °C and 120 V for 3 h. The specificity

GACR grant 301 ⁄ 05 ⁄ 0416. Personnel costs were partly
covered from the research plan No. AVOZ50040507.
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