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DNA modification with cisplatin affects sequence-specificDNA binding of p53 and p73 proteins in a targetsite-dependent mannerHana Pivonˇkova´1, Petr Pecˇinka1, Pavla Cˇesˇkova´2and Miroslav Fojta11 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic2 Masaryk Memorial Cancer Institute, Brno, Czech RepublicThe tumor suppressor protein p53 is known as a tran-scription factor involved in cell cycle control [1–3]. Itplays a crucial role in preventing malignant transfor-mation of a cell via induction of cell cycle arrest orprogrammed cell death in response to stress conditions(e.g. DNA damage). The functions of p53 are closelyrelated to sequence-specific recognition of response ele-ments [p53 DNA-binding sites (p53DBSs)] in promot-ers of downstream genes such as p21WAF1 ⁄ CIP1(involved in cell cycle arrest), Bax (apoptosis), andmdm2 (negative feedback regulation of p53) [1–3].Using chromatin immunoprecipitation combined witha paired-end ditag DNA sequencing strategy, Weiet al. have recently established a global map of p53-binding sites encompassing over 540 loci in the humangenome [4]. A typical p53DBS consists of two tandemcopies of the motif RRRCWWGYYY (where R ¼ Aor G, Y ¼ C or T, and W ¼ A or T), which may beseparated by one or more base pairs [4,5]. Natural p53response elements exhibit surprisingly high sequencevariability and may contain one or several nucleotidesnot fitting the above formula [6,7]. The p53 proteinbinds to the response elements as a tetramer via itscore domain. The importance of p53 sequence-specificKeywordscisplatin; DNA damage; protein p73;sequence-specific DNA recognition; tumorsuppressor protein p53CorrespondenceM. Fojta, Institute of Biophysics, Academyof Sciences of the Czech Republic,Kra´lovopolska´135, CZ-612 65 Brno,Czech RepublicFax: +420 541211293Tel: +420 541517197E-mail: fojta@ibp.cz(Received 25 April 2006, revised 18 July2006, accepted 17 August 2006)doi:10.1111/j.1742-4658.2006.05472.xProteins p53 and p73 act as transcription factors in cell cycle control, regu-lation of cell development and ⁄ or in apoptotic pathways. Both proteinsbind to response elements (p53 DNA-binding sites), typically consisting oftwo copies of a motif RRRCWWGYYY. It has been demonstrated previ-ously that DNA modification with the antitumor drug cisplatin inhibitsp53 binding to a synthetic p53 DNA-binding site. Here we demonstratethat the effects of global DNA modification with cisplatin on binding ofthe p53 or p73 proteins to various p53 DNA-binding sites differed signifi-cantly, depending on the nucleotide sequence of the given target site. Therelative sensitivities of protein–DNA binding to cisplatin DNA treatmentcorrelated with the occurrence of sequence motifs forming stable bifunc-tional adducts with the drug (namely, GG and AG doublets) within thetarget sites. Binding of both proteins to mutated p53 DNA-binding sitesfrom which these motifs had been eliminated was only negligibly affectedby cisplatin treatment, suggesting that formation of the cisplatin adductswithin the target sites was primarily responsible for inhibition of the p53 orp73 sequence-specific DNA binding. Distinct effects of cisplatin DNAmodification on the recognition of different response elements by the p53family proteins may have impacts on regulation pathways in cisplatin-treated cells.AbbreviationscisPt-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 4693DNA binding is underlined by the fact that most ofthe cancer-related point mutations of p53 are locatedin its core domain and the mutants are typicallyunable to recognize the p53 response elements [1,8,9].Besides the nucleotide sequence, binding of p53 tothe p53DBSs appears to depend on conformationalfeatures of its target sites. It has been proposed thatintrinsic bending of the p53DBSs contributes signifi-cantly to the stability of the p53–DNA complexes [10].In addition, interactions of the p53 protein with cer-tain response elements can be controlled by changes inDNA topology inducing formation of non-B DNAstructures within the binding sites [11,12]. Interactionsof p53 with DNA are regulated mainly via post-trans-lational modifications (phosphorylation, acetylation)within the protein C-terminal domain [3,13,14]. Trun-cated forms of p53 lacking a negative-regulating seg-ment at the protein C-terminus (residues 369–383 [15])are constitutively active for sequence-specific DNAbinding [7,16]. On the other hand, the C-terminus ofp53 was shown to be critical for its conformation-selective DNA binding [11,12,17,18] and to favor p53interactions with p53DBSs within long DNA molecules[19,20].The p73 protein has been identified as a p53 homo-log exhibiting 63% amino acid sequence identity in theDNA-binding domain [21–23]. In agreement with thishomology, the p73 protein can recognize the sameresponse elements as the p53 protein and activate ananalogous set of downstream genes. Multiple spliceisoforms of the p73 protein have been found that differin the structure of their N-terminal and ⁄ or C-terminaldomains [21,22]. Although it was originally supposedthat the p53 homologs have redundant functions in theregulation of gene expression, more recent data suggestthat p73 and p63 proteins do not act as ‘classic’ tumorsuppressors, but rather play important roles in theregulation of cell development and differentiation[21,23]. Nevertheless, some observations suggest thatp73 is involved in the cellular response to DNA dam-age and in apoptosis control [24,25].Cisplatin [cis-diamminedichloroplatinum(II)] is aclinically used anticancer agent [26,27]. The drug bindscovalently 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 globallymodified chromosomal DNA comprises about 50%of 1,2-GG IACs, 25% of 1,2-AG IACs, 10% of1,3-GNG IACs and interstrand crosslinks, and another2–3% of monofunctional adducts. It has been foundthat cisplatin cytotoxicity is related mainly to the IACsthat induce significant changes in the DNAconformation, including bending and unwinding of theDNA double helix [26,28]. The lesions are selectivelybound by a variety of nuclear proteins, and it was pro-posed that these interactions are important for theanticancer activity of the drug [26,29,30].Interactions of the p53 protein with cisplatin-modi-fied DNA (cisPt-DNA) have recently been studied [31–36]. In the absence of the p53DBS, enhancement ofp53 sequence-nonspecific DNA binding due to DNAcis-platination was observed [33–36]. On the otherhand, the same DNA treatment resulted in inhibitionof p53 sequence-specific binding [31,32]. An analogousinhibitory effect was observed with the anticancer tri-nuclear platinum complex BBR3464 but not with theclinically ineffective transplatin. Quite recently, it hasbeen shown that DNA modification with a transplatinanalog, trans-[PtCl2NH3(4-hydroxymethylpyridine)],inhibits p53 binding to the same p53DBS similarly asdoes cisplatin [37]. It has been proposed that the inhib-itory effects of the anticancer platinum complexes aredue to the formation of platinum adducts within thep53DBS [31,32]. To our knowledge, no analogousstudies of the p73 protein interactions with chemicallydamaged DNA have been reported yet.In this work, we investigated the effects of globalDNA modification with cisplatin on sequence-specificbinding of p53 and p73 proteins to different targetsites. We demonstrated that the sensitivity of the pro-tein–DNA interactions to cisplatin DNA treatmentcorrelated with the occurrence of sequence motifsforming the cisplatin IACs (namely GG and AG dou-blets) within the given p53DBS. Binding of both pro-teins to mutated target sites not containing thesemotifs was not significantly affected by the DNA cis-platination. Formation of the cisplatin adducts outsidethe p53DBSs did not apparently influence p53sequence-specific DNA binding.ResultsTo analyze the sequence-specific DNA binding of p53and p73 proteins, we designed 50-mer oligonucleotidesubstrates bearing various p53DBSs (Fig. 1). In mostexperiments, we used a C-terminally truncated, consti-tutively active p53(1–363) to eliminate the sequence-nonspecific p53 interactions with the cis-platinatedDNA, 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 the32P-labeled 50-mer targets resultedin the appearance of a distinct retarded band R53inthe polyacrylamide gel (Fig. 2). Binding of the p73bCisplatin effects on p53p73 DNA recognition H. Pivonˇkova´et al.4694 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBSFig. 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-mids). The left part of the scheme shows two p53DBSs derived from natural p53 response elements in p21 (5¢-promoter) and mdm2promoters, as well as the synthetic p53DBS PGM1. Motifs forming bifunctional adducts with cisplatin are highlighted (GG doublets are inbold and underlined, AG doublets are in bold, and GNG triplets are marked by brackets). The p53DBSs shown on the right are derivatives ofp21 (p21a and p21b) or pPGM1 (pPGM4). In the latter targets, the incidence of the cisplatin-reactive sites was reduced or eliminated. Basesnot fitting the ‘canonical’ p53DBS [5] are denoted by lower-case letters.ABFig. 2. Electrophoretic mobility shift assay of sequence-specific binding of p53 or p73 proteins to a 50-mer oligonucleotide (oligo) involvingthe p53 DNA-binding sites (p53DBSs). (A) The32P-labeled p21 target was incubated with the given protein in presence of competitor calfthymus DNA, and this was followed by electrophoresis on 5% polyacrylamide gel. Lane 1 contains only DNA without any protein; lanes 2, 3and 4 correspond to DNA complexes with p53(1–363), p73d and p73b, yielding retarded bands R53,R73dand 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 super-shifted bands are denoted as SR53,SR73dand SR73b; the presence of two supershifted bands in each of the lanes 5–7 corresponds to twopossible stoichiometries of the antibody–protein complexes). (B) Sections of an autoradiogram showing retarded bands due to binding ofp53(1–363) or p73d proteins to 50-mer target oligos containing PGM1, PGM4, mdm2, p21, p21a and p21b sites. Other details as in (A), lanes2 and 3.H. Pivonˇkova´et al. Cisplatin effects on p53p73 DNA recognitionFEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4695and p73d proteins to the DNA targets caused the for-mation of analogous retarded bands (denoted as R73bor R73d, respectively; lanes 3 and 4 in Fig. 2A) whosemobilities reflected different molecular weights of thep73 isoforms. To verify the specificity of the bandshifts for DNA complexes with the proteins studied,we used the band supershift assay with antibodiesagainst the p53 or p73 proteins. Addition of the DO-1 antibody [17,38,39] mapping to the N-terminus ofthe p53 protein resulted in further retardation of thespecific p53–DNA complexes (lane 5 in Fig. 2A),producing two supershifted bands (SR53; Fig. 2A).Formation of the two bands corresponded to twopossible stoichiometries of the antibody–p53 complex,involving either one or two antibody molecules boundper p53 tetramer [16,39]. For supershifting of DNAcomplexes with the p73 constructs, which were taggedwith hemagglutinin (HA), we used antibody to HAand obtained analogous band patterns to thoseobtained with p53 (Fig. 2; lanes 6–7, bands SR73band SR73d), confirming the specificity of the observedprotein–DNA complexes. All 50-mer substrates usedin this work were efficiently bound by the p53 andp73 proteins [shown in Fig. 2B for p53(1–363) andp73d], although their affinities for the proteins dif-fered to some extent (which was manifested by differ-ent intensities of the R bands). To eliminate thesedifferences, the effects of DNA cis-platination on theprotein–DNA interactions were always normalizedwith the intensity of the retarded band resulting fromprotein binding to the same but unmodified p53DBS.Effects of cisplatin DNA modification onsequence-specific binding of the p53 proteinPreviously, it has been shown [31,32] that DNA modi-fication with cisplatin causes dose-dependent inhibitionof the full-length (fl) p53 sequence-specific DNA bind-ing to the synthetic target site PGM1 (Fig. 1). Here,we studied the effects of DNA treatment with cisplatinon p53(1–363) binding to the p53DBSs PGM1, p21and mdm2 (Fig. 1) within the 50-mer oligonucleotides(oligos) (Fig. 3A). All targets were treated with thedrug in excess of nonspecific calf thymus DNA. Inter-action of the protein with any of these targets was sig-nificantly affected by the cisplatin treatment, but thelevels of inhibition observed with individual p53DBSsat the same degree of global DNA cis-platination dif-fered significantly. The steepest decrease in p53–DNAbinding with degree of DNA modification was exhib-ited by the mdm2 target. The R53band due to thep53–mdm2 complex exhibited only 10% intensity forrb¼ 0.02, compared to the R53band due to proteinbinding to the same but unmodified substrate (the rbvalue refers to the number of platinum atoms per totalDNA nucleotide). In contrast, the PGM1 and p21 tar-gets retained 75% and 53% of the p53-binding capa-city at rb¼ 0.02, respectively (Fig. 3A). Increasing theDNA modification degree to rb¼ 0.04 resulted in adecrease of p53–p21 binding to 42%, whereas thePGM1 site bound only 16% of the protein, comparedto the same but unmodified p53DBS. At rb¼ 0.06, allmdm2, PGM1 and p21 targets exhibited very weakp53 binding (about 4% for mdm2 and PGM1 and10% for p21).Sensitivity of the sequence-specific p53 DNAbinding to DNA cis-platination depends on theincidence of cisplatin-reactive motifs within thep53DBSsThe mdm2, PGM1 and p21 target sites (Fig. 1) differsignificantly in the occurrence of sequence motifsknown to form the cisplatin IACs [26,27]. The p21 site,showing the weakest sensitivity of p53 binding to cisp-latin treatment, contains only one GGG triplet withinthe p53DBS. The PGM1 site possesses two AGG tri-plets in one strand and two AG steps in the other. Themdm2 target, whose interaction with p53 was moststrongly affected by DNA cis-platination, containsGG, GGG and AG motifs in one strand and GG andGTG motifs in the other, thus offering not only thehighest total number of reactive motifs among thep53DBSs tested, but also the highest number of sitesknown to be modified most frequently (i.e. the GGdoublets).For the subsequent experiments, we designedmutated p53 target sites from which the cisplatin-react-ive motifs were eliminated. Two p53DBSs were derivedfrom the p21 target site (Fig. 1); in p21a, the GGGtriplet in the bottom strand was mutated into GAG.This exchange resulted in elimination of the mostreactive GG doublets and the introduction of lessreactive AG and ⁄ or GNG motifs [26]. In p21b, theGGG triplet in the bottom strand was replaced byGAA, which contains neither RG nor GNG motifs(Fig. 1); owing to this mutation, all sites suitable forformation of the bifunctional cisplatin adducts wereremoved from the p53DBS. In addition, we derivedanother ‘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-specificp53 binding comparable to that of the parent targets(Fig. 2B).Cisplatin effects on p53p73 DNA recognition H. Pivonˇkova´et al.4696 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBSWe studied how the cisplatin treatment influencesinteraction of the p53(1–363) protein with themutated target sites. The 50-mer oligos containingsequences p21a, p21b or PGM4 were treated withcisplatin as above. DNA modification to rb¼ 0.02resulted 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 thep21a and p21 targets appeared at rb¼ 0.04 (35% or58% inhibition, respectively). At rb¼ 0.06, the p21atarget retained 45% of the p53 binding, thus exhibit-ing at least four times higher binding capacity thanthe natural p21 p53DBS treated in the same way.Binding of p53 to the mutated target p21b exhibitedeven more remarkable resistance to the cisplatin treat-ment. For rbvalues 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 ofp21b, showing practically no inhibition of p53–PGM4binding for rb¼ 0.02 or 0.04 and about 10% inhibi-tion for rb¼ 0.06. The PGM4 site also exhibitedpractically no loss of its p53-binding capacity due tothe DNA cis-platination when located within a474 base pair fragment of the pPGM4 plasmid (notshown), in contrast to the behavior of the analogouspPGM1 fragment [31]. These data revealed a clearcorrelation between the sensitivity of the p53sequence-specific DNA binding to DNA treatmentwith cisplatin and the ability of the particular p53target site to accommodate the cisplatin IACs. Thehigher the probability of formation of the cisplatinIACs within the p53DBSs due to the occurrence ofthe GG, AG and ⁄ or GNG motifs, the stronger theinhibition of the p53 sequence-specific DNA bindingto these targets caused by the DNA treatment withcisplatin.ABFig. 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 ofautoradiograms showing the R53bands corresponding to complexes of p53 with the 50-mer target oligonucleotides (oligos) (Fig. 2). Theextents of DNA modification with cisplatin (rb) are indicated. Other details are as in Fig. 2. The graphs show the dependence of relative p53binding to the targets on the degree of DNA modification (data obtained from densitometric tracing of the autoradiograms; for each targetsite, the intensity of the R53band 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. Pivonˇkova´et al. Cisplatin effects on p53p73 DNA recognitionFEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4697We 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 ofthe targets tested (not shown). The effects of thedegree of DNA cis-platination on recognition of theparticular target site by the fl p53 were analogous tothose observed with the C-terminally truncated p53(1–363) (shown in Fig. 4 shown for the mdm2, PGM1and PGM4 targets).Effects of DNA cis-platination on sequence-specific DNA binding of p73 proteinsWe tested the influence of DNA modification withcisplatin on the binding of two p73 isoforms, p73d andp73b, to the p21 target site (Fig. 5). The intensity ofthe resulting R73dband decreased almost linearly withincrease in the cis-platination level; for rb¼ 0.06,about 90% inhibition of p73d–p21 binding wasobserved. Almost the same results were obtained wheninteraction of the p73b isoform with the p21 targetwas examined (Fig. 5). In contrast, modification ofp21b to rb¼ 0.02 or 0.04 had no significant effect onits interaction with either of the p73 isoforms; at rb¼0.06, only slight (10–15%) inhibition of binding wasdetected. Thus, the p73d and p73b proteins exhibitedbehavior upon binding to cisplatin-treated p21 andp21b target sites that was very close to that of the p53protein. Analogous results were obtained with thePGM1 and PGM4 target sites (not shown).Competition experimentsWe studied the influence of cisplatin DNA modifica-tion on the competition between two p53 target sitesfor the protein (Fig. 6). The 474 base pair fragmentsof plasmids pPGM1 or pPGM4 were used as thesequence-specific competitors, and changes in p53(1–363) binding to the32P-labeled 50-mer targets were fol-lowed. We first tested the effect of the presence of thecompetitor 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 R53band intensities due to bindingof a portion of the p53 molecule to the competitorp53DBS. Modification of the pPGM1 fragment withcisplatin caused a reduction of its competitiveness,which was manifested by increasing relative intensityof the R53band yielded by the p53 complex with theradiolabeled PGM1 probe. When the pPGM1 frag-ment was cis-platinated to rb¼ 0.04 or 0.06, its pres-ence had practically no effect on the R53bandintensity, suggesting that the modified pPGM1 frag-ment had lost its ability to compete for the protein(Fig. 6A). In contrast, the competition ability of thepPGM4 fragment was not significantly influenced byits cis-platination. This observation was in agreementwith the resistance of p53 binding to the PGM4 targetsite to the cisplatin DNA treatment (see above).In addition, we modified with cisplatin equimolarmixtures of the pPGM1 fragment with the32P-labeled50-mer targets p21 or p21b (in the presence of nonspe-cific competitor DNA), and performed a p53-bindingassay (Fig. 6B). In the unmodified DNA, the compet-itor pPGM1 fragment caused about 70% inhibition ofp53(1–363) binding to either of the two targets. Modi-fication of the p21 ⁄ pPGM1 mixture resulted inrb-dependent inhibition of p53 binding to the p21 tar-get, but in contrast to the results shown in Fig. 3(where only the p21 target and nonspecific competitorDNA were present in the sample), the cisplatin inhibi-tion effect was detectable only at rb¼ 0.04 and 0.06.The apparent lack of the cisplatin effect at rb¼ 0.02can be attributed to partial loss of the competitivenessof the pPGM1 fragment due to its modification, whichcompensated for inhibition of p53 binding to the (rel-atively less reactive) p21 target. When the mixture ofthe p21b target with the pPGM1 competitor fragmentwas treated with cisplatin in the same way, the inten-sity of the R53band on the autoradiogram increasedwith the degree of DNA modification. The increasewas already significant at rb¼ 0.02. At rb¼ 0.04 or0.06, the relative intensity of the R53band reachedabout 90% of the value observed with unmodifiedDNA (Fig. 6B). Such behavior reflected inhibition ofp53 binding to the competitor pPGM1 fragment dueto its cis-platination, whereas interaction of the proteinwith the p21b target remained practically unaffectedFig. 4. Effects of DNA modification with cisplatin on full-length p53binding to the mdm2, PGM1 and PGM4 targets. For more details,see Figs 2 and 3.Cisplatin effects on p53p73 DNA recognition H. Pivonˇkova´et al.4698 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBSunder the same conditions. The results of these modelcompetition experiments suggest that global modifica-tion of DNA with cisplatin may shift the distributionof the p53 protein among different target sites, depend-ing on the susceptibility of the particular p53DBSs tomodification with the drug.DiscussionIt has been demonstrated previously that interactionsof the tumor suppressor protein p53 with DNA areinfluenced by covalent modification of the DNA byantitumor platinum complexes [31–36]. Sequence-non-specific DNA binding (in the absence of the p53DBS)of the p53 protein was significantly enhanced by DNAmodification with cisplatin [31,33,34]. The ability ofp53 to recognize the cisPt-DNA was more pronouncedfor the post-translationally unmodified (‘latent’) formof the protein than for its ‘activated’ forms [33].Recently, it has been reported that accessibility of thep53 CTDBS is critical for (sequence-nonspecific) cisPt-DNA recognition [34]. On the other hand, sequence-specific binding of p53 to the synthetic p53DBS PGM1was inhibited in cisplatin-treated DNA [31,32]. As thePGM1 site contains several sequence motifs known toform the most abundant cisplatin adducts (see Fig. 1),the cisplatin inhibitory effects could be explained byDNA damage within the p53DBS. It is known that thecisplatin IACs induce considerable DNA bending anduntwisting as well as perturbation of hydrogen bond-ing within the base pairs [26–28]. Cisplatin adductsoccurring within p53DBS can therefore be expected tocause 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 truncatedp53(1–363) proteinIn this work, we studied the effects of cisplatin treat-ment of various p53DBSs on the sequence-specificbinding of a truncated tetrameric p53 construct lackingthe 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 p53CTDBS has been proposed to cooperate with the coredomain in complex p53–DNA interactions. TheCTDBS has been shown to be essential for p53 bind-ing to target sites adopting non-B conformations (suchas stem–loop or cruciform structures) [11,12,45–47].On the other hand, p53 constructs lacking the CTDBSare capable of efficient binding to short linear modelDNA targets in which the p53DBS is present in itsdouble-helical B-form. Moreover, deletion of theCTDBS (amino acids 363–382) makes it possible toseparate sequence-specific p53 DNA binding fromother modes of p53–DNA interaction that are medi-ated by the protein C-terminus, particularly thesequence-nonspecific binding of p53 preferentially tocisPt-DNA [33,34]. Another CTDBS-lacking tetramericp53 construct, p53CT (spanning amino acids 94–360),has recently been used by Weinberg et al. for evalua-tion of the protein-binding affinities for 20 natural p53recognition elements [7]. A comparative study invol-ving four of them showed practically the same cooper-ative binding of the fl p53 as exhibited by p53CT [7].Fig. 5. The effects of DNA modification with cisplatin on binding ofthe 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 2 and 3.H. Pivonˇkova´et al. Cisplatin effects on p53p73 DNA recognitionFEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4699Likewise, our parallel experiments withp53 yieldedresults that were very similar to those obtained withp53(1–363) (Figs 3 and 4). Hence, the C-terminallytruncated constructs are suitable models for comparat-ive studies of p53 sequence-specific DNA binding tovarious and ⁄ or variously modified target sites.Inhibition of p53 sequence-specific DNA bindingis linked to cisplatin adduct formation within thep53DBSs but not outside these target sitesThe 50-mer target DNA substrates were treated withthe drug in the presence of an excess of nonspecificcompetitor calf thymus DNA mimicking random-sequence natural genetic material that can accommo-date the cisplatin adducts regardless of the reactivityof the particular p53DBS. The frequency of DNAmodification within the p53DBSs could thus be expec-ted to reflect the known distribution of cisplatinadducts in globally modified chromosomal (genomic)DNA [26]. Provided that the cisplatin inhibitory effecton p53 sequence-specific DNA binding is linked pri-marily to the IACs formed within the target sites, thesusceptibility of different targets to the drug treatmentshould correlate markedly with the incidence of thecisplatin-reactive motifs in the p53DBSs. Such a corre-lation was indeed found: the sensitivity of the targetsites to treatment with the drug followed the trendmdm2 > PGM1 > p21 > p21a > p21b  PGM4, inaccordance with the number and kind of motifs suit-able for formation of the IACs inside the p53DBSs(Fig. 1).ABFig. 6. Competition between two differentp53 target sites in globally cisplatin-modifiedDNA for the p53(1–363) protein. In (A),32P-labeled, unmodified PGM1 50-mer wasmixed with cisplatin-treated competitor frag-ments of plasmids pPGM1 or pPGM4 (andwith unmodified calf thymus DNA) prior toaddition of the p53 protein. When the com-petitor fragment was unmodified, the p53protein was distributed between it and thelabeled probe target (a). Upon cis-platinationof the pPGM1 competitor fragment (b), itsaffinity for the protein was decreased dueto formation of cisplatin adducts within thep53 DNA-binding site (p53DBS), resulting inincreased p53 binding to the labeled probe.The pPGM4 fragment (c) contains cisplatin-resistant p53DBS, and its cis-platination didnot change its competitiveness for p53(1–363). In (B), the32P-labeled targets p21 (i)and p21b (ii) were treated with cisplatintogether with the competitor pPGM1 frag-ment, and this was followed by the p53-binding assay. Such treatment resulted in adecrease of p53 binding to the p21 target[in agreement with formation of the adductswithin both p21 and pPGM1 p53DBSs; see(i)]. In contrast, apparent p53 binding to thep21b target increased under the same con-ditions [because the cisplatin adducts wereformed within p53DBS of the competitorbut not within the p21b target; see (ii)]. Thegraphs show the relative binding of p53 tothe radiolabeled targets as a function of rb;the intensities of the R53bands observedfor the unmodified targets in the absence ofthe competitor fragments (first samples ofeach set) were taken as 1. For other details,see Figs 2 and 3.Cisplatin effects on p53p73 DNA recognition H. Pivonˇkova´et al.4700 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBSIn the p21 50-mer target and its derivatives p21aand p21b, the 5¢-neighboring guanines in the ‘top’strand form another GG doublet with the first guanineof the p53DBS (Fig. 1). Interestingly, the presence ofthis reactive motif had no conspicuous effect on p53–p21b binding in the cisplatin-treated DNA, as therewere no significant differences between the behavior ofp21b and that of PGM4 (lacking this boundary GGdoublet; Fig. 1). The results presented in this article donot make it possible to decide whether a single cisplat-in IAC, wherever it is within the p53DBS, can fullyabrogate p53 sequence-specific DNA binding, or whe-ther the protein can recognize such a cis-platinatedsite, albeit with lower affinity. Nevertheless, our datashow clearly that a single reactive motif located withinthe 20 base pair recognition element (e.g. in p21;Fig. 1) caused significant sensitivity of p53–p53DBSbinding to the DNA treatment with cisplatin, whereasthe presence of an overlapping GG doublet formed byone guanine inside and the other outside the p53DBSwas practically without effect.Under the conditions used in this work, the appar-ent sensitivity of p53 (or p73) DNA binding to cis-pla-tination was influenced primarily by the probability ofadduct formation within the target sites, regardless ofthe positions of the cisplatin adducts. The adduct posi-tioning may be nevertheless be important with respectto the stereochemistry of the protein–DNA recognition(cis-platination induces significant bending and tor-sional deformations of the DNA double helix [28]) andthe availability of functional groups ensuring the essen-tial protein–DNA contacts. For example, formation ofthe cisplatin crosslinks within the CWWG box (whichrepresents an area where the p53 Arg248 residue inter-acts with the DNA via a minor groove [48]) might beparticularly critical. The mdm2 site is the onlyp53DBS analyzed in this work that involves an AGdoublet within the CWWG tetramer (Fig. 1), whichmay contribute to its high sensitivity to the cisplatintreatment. We tested this possibility using anotherp53DBS containing a single AG doublet (and no otherreactive motif) derived from PGM4 by inverting theTA pair at position 6. Inhibition of p53(1–363) bindingto this site due to its treatment with cisplatin did notexceed the effect observed with the p21a site (alsoinvolving a single AG motif but outside the CWWGbox), suggesting that the highest sensitivity of themdm2 site towards cis-platination was connected withthe abundance of the highly reactive GG motifs ratherthan with the location of the AG doublet within theCWWG tetranucleotide. On the other hand, our pre-liminary results (M. Fojta et al., unpublished data)suggest that the behavior of cisplatin-treated targetsites possessing a single GG motif at various positionsmay differ significantly (more details will be publishedelsewhere).Altered sequence-nonspecific interactions of the p53protein with DNA due to its cis-platination outside thep53DBSs might, in principle, influence recognition ofthe target sites by the protein. Nevertheless, controltests 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 (rb¼ 0.06) calfthymus competitor DNA revealed no apparent effectof the competitor modification. This observation wasin agreement with the recently reported lack of abilityof p53(1–363) to recognize the nonspecific cisPt-DNA[34]. Furthermore, we were interested in whether thepresence of cisplatin adducts within DNA stretchesflanking the p53DBSs affects the ability of p53 to bindthe specific sequence. The flanking segments in all 50-mer substrates used in this work (Fig. 1) contain threemotifs expected to form the 1,2-IACs (one GG andtwo AGs). Another two sets of 50-mer substrates, inwhich the PGM1 or PGM4 sites were flanked by seg-ments either totally lacking the cisplatin-reactive motifsor containing multiple guanine doublets and ⁄ or triplets(Fig. 7), were used to check the influence of cisplatinadducts in the vicinity of p53DBS. Again, the effectsof DNA cis-platination on p53(1–363) binding to thesesubstrates were dependent on the presence of cisplatin-reactive motifs within the p53DBS but not within theflanking stretches (Fig. 7), suggesting that cisplatinadducts outside the binding site (albeit close to it) donot significantly affect sequence-specific DNA recogni-tion. However, it should be emphasized that suchconclusions need not be applicable to the post-translationally unmodified form of fl p53, whichexhibits apparently weaker binding to p53DBS but sig-nificant sequence-nonspecific preferential binding toglobally cis-platinated DNA [31,33,34].Binding of p73 proteins to the recognitionelements is affected by DNA cis-platination in asimilar way to p53 bindingIn agreement with the considerable homology betweenthe p53 and p73 DNA-binding (core) domains, the p73protein 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 similarto the p53 protein with regard to the protein domainstructure as well as molecular size. The p73b isoformdiffers from p73d in its C-terminal domain, which,in p73b, is extended by a stretch coded by exons 11and 12. In neither of the p73 isoforms has anotherH. Pivonˇkova´et al. Cisplatin effects on p53p73 DNA recognitionFEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS 4701DNA-binding site (besides the core domain) analogousto the p53 CTDBS been identified. Our results showedthat both p73d and p73b bound efficiently to all(unmodified) p53DBSs used in this work, and thatcisplatin treatment of p21, p21b (Fig. 5), PGM1 andPGM4 (not shown) affected the p73 sequence-specificDNA binding basically in the same manner asobserved with p53.Possible impacts on gene expressionin cisplatin-treated cellsIt has been well established that modification of DNAwith cisplatin affects fundamental processes such asDNA synthesis and transcription [26]. The bifunctionalcisplatin DNA adducts slow down or block DNA orRNA polymerization and can hamper the initiation ofDNA transcription [49]. Strong differential inhibitionof marker gene expression was observed in cells treatedwith cisplatin [50]. Interestingly, expression of geneswith stronger promoters was strongly inhibited,whereas some genes possessing weaker promoters wereinduced. It was proposed that the strong promoterswere 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-trolled by the p53 family proteins), differing in theoccurrence of the cisplatin-reactive nucleotide sequencemotifs, to cisplatin treatment has been conducted todate.In response to genotoxic stress, the wild-type p53can activate two different response pathways withquite different impacts on the fate of the cell. The firstinvolves cell cycle arrest via p21WAF1 ⁄ CIP1inductionand activation of DNA repair processes that, in gen-eral, confer chemoresistance to cancer cells. The otherpathway leads to programmed cell death through acti-vation of proapoptotic genes such as Bax, PUMA andNoxa [1–3]. The apoptosis trigger is the desired eventin cancer therapy. Despite considerable recent progressin understanding the functions of p53 and its homo-logs, it has not yet been clarified how the checkpointproteins decide which pathway to activate. Partic-ularly, no unambiguous correlation between wild-typep53 expression and cancer cell susceptibility to cisplat-in-induced apoptosis has been established. Althoughsome authors reported a clear p53-dependent apoptot-ic response to cisplatin [51–54], other investigationsrevealed a less distinct link between p53 status and cellsensitivity to cisplatin, or even suggested oppositeeffects [55–57]. Several observations suggest that apop-tosis in cisplatin-treated cells may be regulated viap53- and ⁄ or p73-dependent or -independent pathways[24,56,58]. Hence, the response of a cancer cell to cisp-latin seems to be rather complex, and its relationshipto the status of the p53 family proteins does notappear to be straightforward.The results of our in vitro binding experiments sug-gest that the expression of various p53 downstreamgenes might be differentially affected in the cisplatin-treated cells, due to different susceptibilities of thep53 response elements to modification with the drug.The natural p53DBSs [6,7] differ significantly in thisrespect. Among the 20 response elements recentlycharacterized by Weinberg et al. [7], GADD45 (a genetaking part in DNA repair ) no GG, three AGdoublets) and the p21 5¢-site (a single GGG triplet)Fig. 7. The effect of DNA modification withcisplatin on p53(1–363) binding to p53 DNA-binding sites (p53DBSs) flanked by stret-ches either totally lacking sites reactive tocisplatin (PGM1-AT, PGM4-AT) or involvingmultiple reactive motifs (PGM1-GC,PGM4-GC). The flanking stretches areshown at the top; for the p53DBS,s seeFig. 1. The experimental conditions are as inFigs 2 and 3.Cisplatin effects on p53p73 DNA recognition H. Pivonˇkova´et al.4702 FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS[...]... cisplatin- damaged DNA: a clue to anticancer activity of cisplatin FASEB J 12, 791–799 30 Kasparkova J & Brabec V (1995) Recognition of DNA interstrand cross-links of cis-diamminedichloroplatinum(Ii) and its trans isomer by DNA- binding proteins Biochemistry 34, 12379–12387 31 Kasparkova J, Pospisilova S & Brabec V (2001) Different recognition of DNA modified by antitumor cisplatin and its clinically ineffective... recognition and repair In Progress in Nucleic Acid Research and Molecular Biology (Moldave K, ed.), pp 1–68 Academic Press Inc., San Diego 28 Takahara PM, Rosenzweig AC, Frederick CA & Lippard SJ (1995) Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin Nature 377, 649–652 29 Zlatanova J, Yaneva J & Leuba SH (1998) Proteins that specifically recognize cisplatin- damaged... Ganjavi H, Gee M, Narendran A, Freedman MH & Malkin D (2005) Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin Cancer Gene Ther 12, 397– 406 Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA & Sen S (2004) Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and. .. Brabec V (2003) Recognition of DNA modified by antitumor cisplatin by ‘latent’ and ‘active’ protein p53 Biochem Pharmacol 65, 1305–1316 34 Pivonkova H, Brazdova M, Kasparkova J, Brabec V & Fojta M (2006) Recognition of cisplatin- damaged DNA by p53 protein: critical role of the p53 C-terminal domain Biochem Biophys Res Commun 339, 477–484 35 Wetzel CC & Berberich SJ (1998) DNA binding activities of p53. .. 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 in uenced 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... 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 lgÆmL)1 in the reaction mixture) were modified in the presence of an excess of nonspecific calf... 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... 492–498 25 Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG Jr, Levrero M & Wang JY (1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin- induced DNA damage Nature 399, 806–809 26 Jamieson ER & Lippard SJ (1999) Structure, recognition, and processing of cisplatin -DNA adducts Chem Rev 99, 2467–2498 27 Brabec V (2002) DNA modifications by antitumor platinum and ruthenium compounds:... calf thymus DNA (400 lgÆmL)1) with 27, 54 or 81 lm cisplatin The competitor fragments of plasmids pPGM1 or pPGM4 (35 lgÆmL)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... 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 the ´ ´ monoclonal antibodies, Dr Marie Brazdova for her help with preparation of the p53 proteins, and Professor G Melino . DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner Hana Pivonˇkova´1,. cisplatin inhibits p53 binding to a synthetic p53 DNA- binding site. Here we demonstratethat the effects of global DNA modification with cisplatin on binding
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