DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner ˇ ˇ ˇ ´ ˇ ´ Hana Pivonkova1, Petr Pecinka1, Pavla Ceskova2 and Miroslav Fojta1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Masaryk Memorial Cancer Institute, Brno, Czech Republic Keywords cisplatin; DNA damage; protein p73; sequence-specific DNA recognition; tumor suppressor protein p53 Correspondence M Fojta, Institute of Biophysics, Academy of Sciences of the Czech Republic, ´ ´ Kralovopolska 135, CZ-612 65 Brno, Czech Republic Fax: +420 541211293 Tel: +420 541517197 E-mail: fojta@ibp.cz (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, regulation 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 previously 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 significantly, 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 bifunctional 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 cisplatintreated cells The tumor suppressor protein p53 is known as a transcription factor involved in cell cycle control [1–3] It plays a crucial role in preventing malignant transformation 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 elements [p53 DNA-binding sites (p53DBSs)] in promoters of downstream genes such as p21WAF1 ⁄ CIP1 (involved in cell cycle arrest), Bax (apoptosis), and mdm2 (negative feedback regulation of p53) [1–3] Using chromatin immunoprecipitation combined with a paired-end ditag DNA sequencing strategy, Wei et al have recently established a global map of p53binding sites encompassing over 540 loci in the human genome [4] A typical p53DBS consists of two tandem copies of the motif RRRCWWGYYY (where R ¼ A or G, Y ¼ C or T, and W ¼ A or T), which may be separated by one or more base pairs [4,5] Natural p53 response elements exhibit surprisingly high sequence variability and may contain one or several nucleotides not fitting the above formula [6,7] The p53 protein binds to the response elements as a tetramer via its core domain The importance of p53 sequence-specific 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 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition DNA binding is underlined by the fact that most of the cancer-related point mutations of p53 are located in its core domain and the mutants are typically unable to recognize the p53 response elements [1,8,9] Besides the nucleotide sequence, binding of p53 to the p53DBSs appears to depend on conformational features of its target sites It has been proposed that intrinsic bending of the p53DBSs contributes significantly to the stability of the p53–DNA complexes [10] In addition, interactions of the p53 protein with certain response elements can be controlled by changes in DNA topology inducing formation of non-B DNA structures within the binding sites [11,12] Interactions of p53 with DNA are regulated mainly via post-translational modifications (phosphorylation, acetylation) within the protein C-terminal domain [3,13,14] Truncated forms of p53 lacking a negative-regulating segment at the protein C-terminus (residues 369–383 [15]) are constitutively active for sequence-specific DNA binding [7,16] On the other hand, the C-terminus of p53 was shown to be critical for its conformationselective DNA binding [11,12,17,18] and to favor p53 interactions with p53DBSs within long DNA molecules [19,20] The p73 protein has been identified as a p53 homolog exhibiting 63% amino acid sequence identity in the DNA-binding domain [21–23] In agreement with this homology, the p73 protein can recognize the same response elements as the p53 protein and activate an analogous set of downstream genes Multiple splice 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 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 damage 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 crosslinks (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 4694 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 proposed that these interactions are important for the anticancer activity of the drug [26,29,30] Interactions of the p53 protein with cisplatin-modified DNA (cisPt-DNA) have recently been studied [31– 36] In the absence of the p53DBS, enhancement of p53 sequence-nonspecific DNA binding due to DNA cis-platination was observed [33–36] On the other hand, the same DNA treatment resulted in inhibition of p53 sequence-specific binding [31,32] An analogous inhibitory effect was observed with the anticancer trinuclear platinum complex BBR3464 but not with the clinically ineffective transplatin Quite recently, it has been shown that DNA modification with a transplatin analog, trans-[PtCl2NH3(4-hydroxymethylpyridine)], inhibits p53 binding to the same p53DBS similarly as does cisplatin [37] It has been proposed that the inhibitory effects of the anticancer platinum complexes are due to the formation of platinum adducts within the p53DBS [31,32] To our knowledge, no analogous studies of the p73 protein interactions with chemically damaged DNA have been reported yet In this work, we investigated the effects of global DNA modification with cisplatin on sequence-specific binding of p53 and p73 proteins to different target sites We demonstrated that the sensitivity of the protein–DNA interactions to cisplatin DNA treatment correlated with the occurrence of sequence motifs forming the cisplatin IACs (namely GG and AG doublets) within the given p53DBS Binding of both proteins to mutated target sites not containing these motifs was not significantly affected by the DNA cisplatination 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, constitutively active p53(1–363) to eliminate the sequencenonspecific p53 interactions with the cis-platinated DNA, which have been shown to be mediated primarily by the p53 C-terminal DNA-binding site (CTDBS) [34] In the presence of competitor nonspecific DNA, sequence-specific binding of the p53(1– 363) protein to the 32P-labeled 50-mer targets resulted in the appearance of a distinct retarded band R53 in the polyacrylamide gel (Fig 2) Binding of the p73b FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition Fig Scheme of DNA substrates used in this work All p53 DNA-binding sites (p53DBSs) were placed in the center of 50-mer oligonucleotides (oligos), being flanked with the sequences shown on the top (the same stretches flank the p53DBSs in the pPGM1 and pPGM4 plasmids) The left part of the scheme shows two p53DBSs derived from natural p53 response elements in p21 (5¢-promoter) and mdm2 promoters, as well as the synthetic p53DBS PGM1 Motifs forming bifunctional adducts with cisplatin are highlighted (GG doublets are in bold and underlined, AG doublets are in bold, and GNG triplets are marked by brackets) The p53DBSs shown on the right are derivatives of p21 (p21a and p21b) or pPGM1 (pPGM4) In the latter targets, the incidence of the cisplatin-reactive sites was reduced or eliminated Bases not fitting the ‘canonical’ p53DBS [5] are denoted by lower-case letters A B Fig Electrophoretic mobility shift assay of sequence-specific binding of p53 or p73 proteins to a 50-mer oligonucleotide (oligo) involving the p53 DNA-binding sites (p53DBSs) (A) The 32P-labeled p21 target was incubated with the given protein in presence of competitor calf thymus DNA, and this was followed by electrophoresis on 5% polyacrylamide gel Lane contains only DNA without any protein; lanes 2, and correspond to DNA complexes with p53(1–363), p73d and p73b, yielding retarded bands R53, R73d and R73b, respectively In lanes 5–7, the protein–DNA complexes are supershifted with monoclonal antibodies DO-1 (p53) or anti-HA (both p73 isoforms; the respective supershifted bands are denoted as SR53, SR73d and SR73b; the presence of two supershifted bands in each of the lanes 5–7 corresponds to two possible stoichiometries of the antibody–protein complexes) (B) Sections of an autoradiogram showing retarded bands due to binding of p53(1–363) or p73d proteins to 50-mer target oligos containing PGM1, PGM4, mdm2, p21, p21a and p21b sites Other details as in (A), lanes and FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4695 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition and p73d proteins to the DNA targets caused the formation of analogous retarded bands (denoted as R73b or R73d, respectively; lanes and 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 DO1 antibody [17,38,39] mapping to the N-terminus of the p53 protein resulted in further retardation of the specific p53–DNA complexes (lane in Fig 2A), producing two supershifted bands (SR53; 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 obtained with p53 (Fig 2; lanes 6–7, bands SR73b and SR73d), confirming the specificity of the observed protein–DNA complexes All 50-mer substrates used in this work were efficiently bound by the p53 and p73 proteins [shown in Fig 2B for p53(1–363) and p73d], although their affinities for the proteins differed to some extent (which was manifested by different intensities of the R bands) To eliminate these differences, the effects of DNA cis-platination on the protein–DNA interactions were always normalized with the intensity of the retarded band resulting from protein binding to the same but unmodified p53DBS Effects of cisplatin DNA modification on sequence-specific binding of the p53 protein Previously, it has been shown [31,32] that DNA modification with cisplatin causes dose-dependent inhibition of the full-length (fl) p53 sequence-specific DNA binding to the synthetic target site PGM1 (Fig 1) Here, we studied the effects of DNA treatment with cisplatin on p53(1–363) binding to the p53DBSs PGM1, p21 and mdm2 (Fig 1) within the 50-mer oligonucleotides (oligos) (Fig 3A) All targets were treated with the drug in excess of nonspecific calf thymus DNA Interaction of the protein with any of these targets was significantly affected by the cisplatin treatment, but the levels of inhibition observed with individual p53DBSs at the same degree of global DNA cis-platination differed significantly The steepest decrease in p53–DNA binding with degree of DNA modification was exhibited by the mdm2 target The R53 band due to the p53–mdm2 complex exhibited only 10% intensity for rb ¼ 0.02, compared to the R53 band due to protein 4696 binding to the same but unmodified substrate (the rb value refers to the number of platinum atoms per total DNA nucleotide) In contrast, the PGM1 and p21 targets retained 75% and 53% of the p53-binding capacity at rb ¼ 0.02, respectively (Fig 3A) Increasing the DNA modification degree to rb 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 rb ¼ 0.06, all mdm2, PGM1 and p21 targets exhibited very weak p53 binding (about 4% for mdm2 and PGM1 and 10% for p21) Sensitivity of the sequence-specific p53 DNA binding to DNA cis-platination depends on the incidence of cisplatin-reactive motifs within the p53DBSs The mdm2, PGM1 and p21 target sites (Fig 1) differ significantly in the occurrence of sequence motifs known to form the cisplatin IACs [26,27] The p21 site, showing the weakest sensitivity of p53 binding to cisplatin treatment, contains only one GGG triplet within the p53DBS The PGM1 site possesses two AGG triplets in one strand and two AG steps in the other The mdm2 target, whose interaction with p53 was most strongly affected by DNA cis-platination, contains GG, GGG and AG motifs in one strand and GG and GTG motifs in the other, thus offering not only the highest total number of reactive motifs among the p53DBSs tested, but also the highest number of sites known to be modified most frequently (i.e the GG doublets) For the subsequent experiments, we designed mutated p53 target sites from which the cisplatin-reactive motifs were eliminated Two p53DBSs were derived from the p21 target site (Fig 1); in p21a, the GGG triplet in the bottom strand was mutated into GAG This exchange resulted in elimination of the most reactive GG doublets and the introduction of less reactive AG and ⁄ or GNG motifs [26] In p21b, the GGG triplet in the bottom strand was replaced by GAA, which contains neither RG nor GNG motifs (Fig 1); owing to this mutation, all sites suitable for 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) FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS ˇ ´ H Pivonkova et al A Cisplatin effects on p53 ⁄ p73 DNA recognition B Fig 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 R53 bands corresponding to complexes of p53 with the 50-mer target oligonucleotides (oligos) (Fig 2) The extents of DNA modification with cisplatin (rb) are indicated Other details are as in Fig 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 R53 band resulting from p53 binding to unmodified DNA was taken as 1.0, and the intensities of bands corresponding to p53 binding to the same but cisplatin-treated substrate were normalized to this) 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 rb ¼ 0.02 resulted in a decrease of p53 binding to the p21a target by about 15%, which represented weaker inhibition than observed with the p21 target (25% decrease; Fig 3) More conspicuous differences between the p21a and p21 targets appeared at rb ¼ 0.04 (35% or 58% inhibition, respectively) At rb ¼ 0.06, the p21a target retained 45% of the p53 binding, thus exhibiting at least four times higher binding capacity than the natural p21 p53DBS treated in the same way Binding of p53 to the mutated target p21b exhibited even more remarkable resistance to the cisplatin treatment For rb values of 0.02, 0.04 or 0.06, 100%, 91% or 85% of the p21b target was bound by the protein, respectively, when compared to the untreated p21b The behavior of the PGM4 site was similar to that of p21b, showing practically no inhibition of p53–PGM4 binding for rb ¼ 0.02 or 0.04 and about 10% inhibition for rb ¼ 0.06 The PGM4 site also exhibited practically no loss of its p53-binding capacity due to the DNA cis-platination when located within a 474 base pair fragment of the pPGM4 plasmid (not shown), in contrast to the behavior of the analogous pPGM1 fragment [31] These data revealed a clear correlation between the sensitivity of the p53 sequence-specific DNA binding to DNA treatment with cisplatin and the ability of the particular p53 target site to accommodate the cisplatin IACs The higher the probability of formation of the cisplatin 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 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4697 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition We also performed parallel experiments with fl p53 (expressed in insect cells) Like p53(1–363), the fl protein 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 particular target site by the fl p53 were analogous to those observed with the C-terminally truncated p53(1– 363) (shown in Fig shown for the mdm2, PGM1 and PGM4 targets) Effects of DNA cis-platination on sequencespecific DNA binding of p73 proteins We tested the influence of DNA modification with cisplatin on the binding of two p73 isoforms, p73d and p73b, to the p21 target site (Fig 5) The intensity of the resulting R73d band decreased almost linearly with increase in the cis-platination level; for rb ¼ 0.06, about 90% inhibition of p73d–p21 binding was observed Almost the same results were obtained when interaction of the p73b isoform with the p21 target was examined (Fig 5) In contrast, modification of p21b to rb ¼ 0.02 or 0.04 had no significant effect on its interaction with either of the p73 isoforms; at rb ¼ 0.06, only slight (10–15%) inhibition of binding was detected Thus, the p73d and p73b proteins exhibited behavior upon binding to cisplatin-treated p21 and p21b target sites that was very close to that of the p53 protein Analogous results were obtained with the PGM1 and PGM4 target sites (not shown) Competition experiments We studied the influence of cisplatin DNA modification on the competition between two p53 target sites for the protein (Fig 6) The 474 base pair fragments Fig Effects of DNA modification with cisplatin on full-length p53 binding to the mdm2, PGM1 and PGM4 targets For more details, see Figs and 4698 of plasmids pPGM1 or pPGM4 were used as the sequence-specific competitors, and changes in p53(1– 363) binding to the 32P-labeled 50-mer targets were followed We first tested the effect of the presence of the competitor fragments (unmodified or treated with cisplatin) on p53 binding to the unmodified PGM1 probe (Fig 6A) Addition of either of the unmodified fragments (70 ng per sample) resulted in a partial decrease (by 35–45%) of the R53 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 R53 band yielded by the p53 complex with the radiolabeled PGM1 probe When the pPGM1 fragment was cis-platinated to rb ¼ 0.04 or 0.06, its presence had practically no effect on the R53 band intensity, suggesting that the modified pPGM1 fragment had lost its ability to compete for the protein (Fig 6A) In contrast, the competition ability of the pPGM4 fragment was not significantly influenced by its cis-platination This observation was in agreement with the resistance of p53 binding to the PGM4 target site to the cisplatin DNA treatment (see above) In addition, we modified with cisplatin equimolar mixtures of the pPGM1 fragment with the 32P-labeled 50-mer targets p21 or p21b (in the presence of nonspecific competitor DNA), and performed a p53-binding assay (Fig 6B) In the unmodified DNA, the competitor pPGM1 fragment caused about 70% inhibition of p53(1–363) binding to either of the two targets Modification of the p21 ⁄ pPGM1 mixture resulted in rb-dependent inhibition of p53 binding to the p21 target, but in contrast to the results shown in Fig (where only the p21 target and nonspecific competitor DNA were present in the sample), the cisplatin inhibition effect was detectable only at rb ¼ 0.04 and 0.06 The apparent lack of the cisplatin effect at rb 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 (relatively 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 intensity of the R53 band on the autoradiogram increased with the degree of DNA modification The increase was already significant at rb ¼ 0.02 At rb ¼ 0.04 or 0.06, the relative intensity of the R53 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 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition of the protein than for its ‘activated’ forms [33] Recently, it has been reported that accessibility of the p53 CTDBS is critical for (sequence-nonspecific) cisPtDNA recognition [34] On the other hand, sequencespecific binding of p53 to the synthetic p53DBS PGM1 was inhibited in cisplatin-treated DNA [31,32] As the PGM1 site contains several sequence motifs known to form the most abundant cisplatin adducts (see Fig 1), the cisplatin inhibitory effects could be explained by DNA damage within the p53DBS It is known that the cisplatin IACs induce considerable DNA bending and untwisting as well as perturbation of hydrogen bonding within the base pairs [26–28] Cisplatin adducts occurring within p53DBS can therefore be expected to cause severe deformations of the binding site with concomitant destabilization of the p53–DNA interaction (or even prevention of target recognition by the protein) DNA binding of the C-terminally truncated p53(1–363) protein Fig The effects of DNA modification with cisplatin on binding of the p73 proteins to the p21 and p21b target sites In the graph, squares correspond to p73b and triangles to p73d For other details, see Figs and under the same conditions The results of these model competition experiments suggest that global modification of DNA with cisplatin may shift the distribution of the p53 protein among different target sites, depending on the susceptibility of the particular p53DBSs to modification with the drug Discussion It has been demonstrated previously that interactions of the tumor suppressor protein p53 with DNA are influenced by covalent modification of the DNA by antitumor platinum complexes [31–36] Sequence-nonspecific DNA binding (in the absence of the p53DBS) of the p53 protein was significantly enhanced by DNA modification with cisplatin [31,33,34] The ability of p53 to recognize the cisPt-DNA was more pronounced for the post-translationally unmodified (‘latent’) form In this work, we studied the effects of cisplatin treatment 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 constitutively active for sequence-specific DNA binding [16] Models of p53 latency considering the (post-translationally unmodified) p53 C-terminus solely as a negative 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 binding 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 other modes of p53–DNA interaction that are mediated by the protein C-terminus, particularly the sequence-nonspecific binding of p53 preferentially to cisPt-DNA [33,34] Another CTDBS-lacking tetrameric p53 construct, p53CT (spanning amino acids 94–360), has recently been used by Weinberg et al for evaluation of the protein-binding affinities for 20 natural p53 recognition elements [7] A comparative study involving four of them showed practically the same cooperative binding of the fl p53 as exhibited by p53CT [7] FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4699 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition A B Likewise, our parallel experiments with fl p53 yielded results that were very similar to those obtained with p53(1–363) (Figs and 4) Hence, the C-terminally truncated constructs are suitable models for comparative studies of p53 sequence-specific DNA binding to various and ⁄ or variously modified target sites Inhibition of p53 sequence-specific DNA binding is linked to cisplatin adduct formation within the p53DBSs but not outside these target sites The 50-mer target DNA substrates were treated with the drug in the presence of an excess of nonspecific competitor calf thymus DNA mimicking randomsequence natural genetic material that can accommodate the cisplatin adducts regardless of the reactivity 4700 Fig 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 fragments of plasmids pPGM1 or pPGM4 (and with unmodified calf thymus DNA) prior to addition of the p53 protein When the competitor fragment was unmodified, the p53 protein was distributed between it and the labeled probe target (a) Upon cis-platination of the pPGM1 competitor fragment (b), its affinity for the protein was decreased due to formation of cisplatin adducts within the p53 DNA-binding site (p53DBS), resulting in increased p53 binding to the labeled probe The pPGM4 fragment (c) contains cisplatinresistant p53DBS, and its cis-platination did not change its competitiveness for p53(1– 363) In (B), the 32P-labeled targets p21 (i) and p21b (ii) were treated with cisplatin together with the competitor pPGM1 fragment, and this was followed by the p53binding assay Such treatment resulted in a decrease of p53 binding to the p21 target [in agreement with formation of the adducts within both p21 and pPGM1 p53DBSs; see (i)] In contrast, apparent p53 binding to the p21b target increased under the same conditions [because the cisplatin adducts were formed within p53DBS of the competitor but not within the p21b target; see (ii)] The graphs show the relative binding of p53 to the radiolabeled targets as a function of rb; the intensities of the R53 bands observed for the unmodified targets in the absence of the competitor fragments (first samples of each set) were taken as For other details, see Figs and of the particular p53DBS The frequency of DNA modification within the p53DBSs could thus be expected 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 primarily 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 correlation 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 suitable for formation of the IACs inside the p53DBSs (Fig 1) FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS ˇ ´ H Pivonkova et al 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 not make it possible to decide whether a single cisplatin IAC, wherever it is within the p53DBS, can fully abrogate p53 sequence-specific DNA binding, or whether 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 binding to the DNA treatment with cisplatin, whereas the presence of an overlapping GG doublet formed by one guanine inside and the other outside the p53DBS was practically without effect Under the conditions used in this work, the apparent sensitivity of p53 (or p73) DNA binding to cis-platination was influenced primarily by the probability of adduct formation within the target sites, regardless of the positions of the cisplatin adducts The adduct positioning may be nevertheless be important with respect to the stereochemistry of the protein–DNA recognition (cis-platination induces significant bending and torsional deformations of the DNA double helix [28]) and the availability of functional groups ensuring the essential protein–DNA contacts For example, formation of the cisplatin crosslinks within the CWWG box (which represents an area where the p53 Arg248 residue interacts with the DNA via a minor groove [48]) might be particularly critical The mdm2 site is the only p53DBS analyzed in this work that involves an AG doublet within the CWWG tetramer (Fig 1), which may contribute to its high sensitivity to the cisplatin treatment We tested this possibility using another p53DBS containing a single AG doublet (and no other reactive motif) derived from PGM4 by inverting the TA pair at position Inhibition of p53(1–363) binding to this site due to its treatment with cisplatin did not exceed the effect observed with the p21a site (also involving a single AG motif but outside the CWWG box), suggesting that the highest sensitivity of the 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 preliminary results (M Fojta et al., unpublished data) suggest that the behavior of cisplatin-treated target Cisplatin effects on p53 ⁄ p73 DNA recognition 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 unmodified PGM1, PGM4, p21 and p21b targets in the presence of unmodified or cis-platinated (rb 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 50mer 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 segments either totally lacking the cisplatin-reactive motifs or containing multiple guanine doublets and ⁄ or triplets (Fig 7), were used to check the influence of cisplatin adducts in the vicinity of p53DBS Again, the effects of DNA cis-platination on p53(1–363) binding to these substrates were dependent on the presence of cisplatinreactive motifs within the p53DBS but not within the flanking stretches (Fig 7), suggesting that cisplatin adducts outside the binding site (albeit close to it) not significantly affect sequence-specific DNA recognition However, it should be emphasized that such conclusions need not be applicable to the posttranslationally unmodified form of fl p53, which exhibits apparently weaker binding to p53DBS but significant sequence-nonspecific preferential binding to globally cis-platinated DNA [31,33,34] Binding of p73 proteins to the recognition elements is affected by DNA cis-platination in a similar way to p53 binding In agreement with the considerable homology between the p53 and p73 DNA-binding (core) domains, the p73 protein can bind to the p53 response elements [21,22] Among the known p73 splice isoforms [21,23], p73d (coded by exons 2–10 of the p73 gene) is most similar to the p53 protein with regard to the protein domain structure as well as molecular size The p73b isoform differs from p73d in its C-terminal domain, which, in p73b, is extended by a stretch coded by exons 11 and 12 In neither of the p73 isoforms has another FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4701 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition Fig The effect of DNA modification with cisplatin on p53(1–363) binding to p53 DNAbinding sites (p53DBSs) flanked by stretches 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 The experimental conditions are as in Figs and 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 therefore more easily modified by the drug [50] However, to our knowledge, no systematic study of the sensitivity of various promoters (and particularly those controlled by the p53 family proteins), differing in the occurrence of the cisplatin-reactive nucleotide sequence motifs, to cisplatin treatment has been conducted to date In response to genotoxic stress, the wild-type p53 can activate two different response pathways with quite different impacts on the fate of the cell The first 4702 involves cell cycle arrest via p21WAF1 ⁄ CIP1 induction and activation of DNA repair processes that, in general, confer chemoresistance to cancer cells The other pathway leads to programmed cell death through activation of proapoptotic genes such as Bax, PUMA and Noxa [1–3] The apoptosis trigger is the desired event in cancer therapy Despite considerable recent progress in understanding the functions of p53 and its homologs, it has not yet been clarified how the checkpoint proteins decide which pathway to activate Particularly, no unambiguous correlation between wild-type p53 expression and cancer cell susceptibility to cisplatin-induced apoptosis has been established Although some authors reported a clear p53-dependent apoptotic response to cisplatin [51–54], other investigations revealed a less distinct link between p53 status and cell sensitivity to cisplatin, or even suggested opposite effects [55–57] Several observations suggest that apoptosis in cisplatin-treated cells may be regulated via p53- and ⁄ or p73-dependent or -independent pathways [24,56,58] Hence, the response of a cancer cell to cisplatin 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 suggest that the expression of various p53 downstream genes might be differentially affected in the cisplatintreated 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) FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition appear to be the most ‘cisplatin-resistant’, whereas the most ‘reactive’ are the two apoptosis-related response elements Noxa and PUMA BS2 (four GG and two AG, or five GG and one AG, respectively) Global DNA cis-platination may shift the distribution of p53 protein molecules bound to various p53DBSs towards those less susceptible to the modification (Fig 6) However, promoters of functionally related genes (arrest ⁄ repair or apoptosis) not generally tend to cluster along the scale of potential reactivity to cisplatin [6,7] Expression of the p53 downstream effectors in cisplatin-treated cells may thus be entirely unbalanced rather than shifted towards one of the response pathways The generally unpredictable cellular response to cisplatin treatment can be connected with the facts that modification of particular sites in the genome of living cells is naturally stochastic on the one hand, and influenced by the actual structural and functional state of the given chromatin domain on the other Moreover, some of the p53 downstream genes possess more than one p53 response element, and these may differ in their susceptibilities to cis-platination (for example, the second p21 site involves one GG and four AG internal motifs [7]) Cellular response pathways involve many factors whose functions are differentially affected by DNA damage, and regulation of the tumor suppressor protein activity itself can also be influenced by genomic DNA cis-platination, due to preferential binding of the post-translationally unmodified (‘latent’) form of p53 to the cisPt-DNA in the absence of the p53DBS [33–35] Inhibition of sequence-specific p53 (or p73) protein DNA binding due to formation of the cisplatin adducts within its response elements could thus represent an important, but not the only, factor affecting cellular regulation pathways in cells exposed to the drug Genomics and nucleotide triphosphates by Sigma (St Louis, MO, USA) Experimental procedures DNA-binding assays DNA samples Synthetic 50-mer oligonucleotides containing different p53DBSs (Fig 1) were supplied by VBC Genomics (Vienna, Austria) Plasmids pPGM1 and pPGM4 [derivatives 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, Hilden, Germany) Restriction endonucleases were supplied by Takara (Otsu, Japan), thermostable Pfu DNA polymerase by Promega (Madison, WI, USA), PCR primers by VBC DNA modification with cisplatin DNA samples were incubated with cisplatin (Sigma) in 10 mm NaClO4 at 37 °C for 48 h in the dark Radioactively (32P) labeled 50-mer substrates (10 lgỈmL)1 in the reaction mixture) were modified in the presence of an excess of nonspecific calf thymus DNA (400 lgỈmL)1) with 27, 54 or 81 lm cisplatin The competitor fragments of plasmids pPGM1 or pPGM4 (35 lgỈmL)1) were treated in the absence of the calf thymus DNA (the fragments themselves contain random-sequence stretches representing major parts of the DNA molecules), and cisplatin concentrations of 2.3, 4.6 or 6.9 lm were applied to maintain the cisplatin ⁄ nucleotide ratios and thus the levels of global DNA modification The number of cisplatin moieties bound per DNA nucleotide (rb) under controlled conditions was previously determined by flameless atomic absorption spectroscopy or polarographically [31,59] The rb values attained under the conditions used in this work were 0.02, 0.04 or 0.06 Preparation of p53 and p73 proteins C-terminally truncated p53(1–363) and fl p53 were expressed in bacterial Escherichia coli BL21 ⁄ DE3 cells or in insect Sf9 cells, respectively, purified and characterized as described previously [18,33] Proteins p73b and p73d were prepared using the TNTÒ Quick Coupled Transcription ⁄ Translation System (Promega) Plasmids pcDNA3HA-p73b or pcDNA3-HA-p73d (1 lg) coding the respective p73 isoforms (both HA-tagged at their N-termini) were mixed with lL of mm methionine, 40 lL of TNTÒ T7 Quick Master Mix and nuclease-free water to a final volume of 50 lL Samples were incubated at 30 °C for 90 Protein concentrations were determined densitometrically from Coomassie blue G-250-stained polyacrylamide gels In all experiments, the p53 or p73 proteins were mixed with the DNA substrates in mm dithiothreitol, 50 mm KCl, mm Tris (pH 7.6) and 0.01% Triton X-100 (total volume 20 lL) and incubated on ice for 30 The protein ⁄ DNA target site molar ratio (i.e protein tetramers per radiolabeled 50-mer probe) was ⁄ The reaction mixture contained 50 ng of the 32P-labeled oligo and lg of nonspecific competitor 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 H3BO3, 0.7 mm EDTA buffer (pH 8.0) at °C and 120 V for h The specificity of the protein–DNA complexes was checked by a band FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4703 ˇ ´ H Pivonkova et al Cisplatin effects on p53 ⁄ p73 DNA recognition supershifting assay with antibodies DO-1 [38] or anti-HA (Sigma) Gels were dried and autoradiographed using Phosphorimager Storm Band intensities on the gels were quantified with image-quant software Average values and standard errors shown in the graphs were calculated from three experiments Acknowledgements ˇ The authors thank Dr Borˇ ek Vojtesˇ ek for providing 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PGM4 targets) Effects of DNA cis-platination on sequencespecific DNA binding of p73 proteins We tested the in? ??uence of DNA modification with cisplatin on the binding of two p73 isoforms, p73d and p73b,... DNA binding to DNA treatment with cisplatin and the ability of the particular p53 target site to accommodate the cisplatin IACs The higher the probability of formation of the cisplatin IACs within... of cisplatin adducts within the p53 DNA- binding site (p53DBS), resulting in increased p53 binding to the labeled probe The pPGM4 fragment (c) contains cisplatinresistant p53DBS, and its cis-platination