Báo cáo khóa học: Sequence selective binding of bis-daunorubicin WP631 to DNA doc

Sequence selective binding of bis-daunorubicin WP631 to DNA Keith R. Fox 1 , Richard Webster 1 , Robin J. Phelps 1 , Izabela Fokt 2 and Waldemar Priebe 2 1 School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, UK; 2 The University of Texas MD Anderson Cancer Center, Houston, TX, USA We have used footprinting techniques on a wide range of natural a nd synthetic footprinting substrates to examine t he sequence-selective i nteraction of the bis-daunorubicin anti- biotic WP631 with DNA. The ligand produces clear DNase I footprints that are very diff erent f rom t hose s een with other anthracycline antibiotics such as daunorubicin and nogalamycin. Footprints are found in a diverse range of sequences, many of which are rich in G T ( AC) o r G A ( TC) residues. As expected, the ligand binds well to the sequences CGTACG and CGATCG, but clear footprints are also found at h exanucleotide sequences s uch GCATGC and GCTAGC. The various footprints do not contain any par- ticular unique d i-, tri- or tetranucleotide s equences, but are frequently contain the sequence (G/C)(A/T)(A/T)(G/C). All sequences with this composition a re protected by the ligand, thoughitcanalsobindtosomesitesthatdifferfromthis consensus by one bas e pair. Keywords: WP631; anthracycline antibiotic; daunorubicin; footprinting; sequence r ecognition. A large number of ligands are known to b ind to DNA, a nd several o f t hese are important therapeutic agents, partic- ularly in the treatment of cancer. H owever most such agents have little or no sequence selectivity and are therefore extremely cytotoxic and affect a ll rapidly dividing cells. O ne goal for cancer chemotherapy is therefore to produce compounds that only interact with specific genes, or gene products. T he deciphering of many complete genomes gives new impetus to the search for molecules that interfere with the activity of individual genes. Examples o f strategies aimed at realizing this goal i ncluded the fo rmation of intermolecular triplex helices [1,2] a nd the pyrrole-imidazole polyamines [3,4]. The interaction of many small molecules with DNA has been well characterized and several of these have limited sequence recognition p roperties. However, with the exception of the polyamides [3,4] these compound s only r ecognize between two and four base pairs. One means of i ncreasing the selectivity is to produce o ligo- mers of known agents, thereby increasing the binding site size, the selectivity and the strength of binding [5–7]. The first examples of such agents included t he bis-intercalat- ing acridines. These generally bind more stron gly than simple mono-intercalators, though t his rarely approaches the theoretical limit of the square of the binding constant, because of conformational and structural restrictions imposed by the linkers between the two intercalators. In addition, because the parent compounds bind to almost all DNA sequences, t he oligomers show little o r n o s equence selectivity. The anthracycline a ntibiotics are well known antitumour agents [8–11] a nd, although they d isplay a p leiotropic mechanism of action, DNA is their p rimary cellular target. The best characterized members of this group are dauno- rubicin (daunomycin) and doxorubicin ( adriamycin). These agents bind to DNA by intercalation, with the amino sugar daunosamine positioned i n the DNA minor groove. Several crystal s tructures h ave been reported f or the interaction of these ligands with oligonucleotides, i ncluding CGTACG [12,13], CGATCG [14,15] a nd TGTACA and T GATCA [16]. They possess some sequence specificity and high resolution footprinting has suggested that they bind best to the sequences 5 ¢-(A/T)CG and 5¢-(A/T)GC [1 7–19]. There have been a number of attempts to produce bis- intercalating dau norubicin derivatives, with increased affin- ity for DNA. I n early studies these were linked through C 13 and C 14 as these are chemically accessible [20,21]. H owever these positions are involved in DNA binding and the modifications decreased the affinity of each mono mer. More recently dimers of daunorubicin have been produced by linking between t he C-4¢ or C-3¢ sugar positions [22,23]. These compounds were designed after examination of the crystal structure of daunorubicin bound to CGTACG [13]. This s tructure c ontains two daunorubicin molecules which are intercalated at the CpG steps with their amino sugars facing each other at the centre o f the complex, with the 3¢-amines s eparated by 6 –7 A ˚ .Ap-xylyl linker w as chosen to link the two h alves of the dimer generating WP631 (Fig. 1), linked at the 3¢-positions and WP652 (linked at the modified 4¢-positions). These compounds show promising biological activity and a re significantly more cyt otoxic than doxorubicin a gainst multi-drug resistant tumours [22,23]. WP631 has been shown t o be a n Sp1 site-specific drug [24], an a ctivator o f nuclea r fa ctor-j B [25], a nd an inhibitor of Tat transcription in HIV [26], as well as a general antiproliferative agent. Spectroscopic methods have been u sed to examine the binding of WP631 to DNA [27]. Continuous variation Correspondence to K. R. Fox, Schoo l of B iological Sciences, U niver- sity of Southampton, Bassett Crescent East, South ampton SO16 7PX, UK. Fax: +4 4 23 80594459; Tel.: +44 23 80594374; E-mail: K.R.Fox@soton.ac.uk Abbreviations: DEPC, dieth ylpyrocarbonate. (Received 4 June 2 004, r evised 14 July 2004, accepted 15 July 2004) Eur. J. Biochem. 271, 3556–3566 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04292.x analysis revealed up to six distinct binding modes to herring sperm DNA. T h e tightest of thes e corresponded to the i nteraction of one drug molecule with six base p airs with an as sociation c onstant of 3 · 10 11 M )1 at 2 0 °C. High resolution melting s tudies showed that the ligand bound preferentially to GC-rich DNA regions [27]. B y comparison with the crystal structures of daunorubicin we would expect these ligands to bind to the s equence CG(A/T)(A/T)CG. NMR [28] a nd cry stal structures [ 29] have been derived f or the interaction of WP631 with CGTACG and CGATCG, respectively, and as expected show that the ligand binds by bisintercalation with each chromophore inserted into the CpG steps, with four base pairs sandwiched between them. In contrast, prolonged incubation of WP652 with CGTACG resulted in precipi- tation, and the NMR structure was d etermined for this ligand bound to TGTACA [28]. In t his structu re the ligand is bound a cross the sequence P yGTPu, with only t wo base pairs between the i ntercalated chromophores. These studies have demonstrated that WP631 binds tightly to DNA by bisintercalation and assume that it recognizes the sequence CG(T/A)(T/A)CG. How ever, there have been no previous studies examining its sequence binding preferen ces, t hough i t has been demonstrated that WP631 inhibits Sp1-activated transcription in vitro [24,30 ]. In this paper we e xamine t he DNA sequence s pecificity o f WP631 using a r ange of footprinting techniques o n several different DNA fragments. Materials and methods Chemicals and enzymes Oligonucleotides for preparing the v arious DN A f ragments were purchased from Oswel DNA service (Southampton, UK). These were stored in water at )20 °C,anddilutedto working concentrations immediately before use. P lasmid pUC19 was purchased from Pharmacia. DNase I was purchased from Sigma and stored at )20 °C at a concen- tration o f 7200 UÆmL )1 . Restriction enzymes and reverse transcriptase w ere purchased from Promega. WP631 (Fig. 1) was prepared as previously described [23]. DNA fragments The sequences of the various fragments used in this work are shown in the various differential plots ( see b elow). TyrT(43–59) is a 110 base pair fragment, which has been widely used in p revious footprinting studies [31]. This labelled DNA fragment was obtained by cutting the plasmid w ith Eco RI and AvaIandwaslabelledatthe 3¢-end of the EcoRI site with [ 32 P]dATP[aP] using reverse transcriptase. Fragments MS1 and MS2 were designed to contain all 136 possible tetranucleotide sequences [32]. These t wo fragments contain the same sequence in opposite orientations, allowing visualization of f ootprints that are located at either e nd. Fragments DMG60Y a nd DMG60R contain oligopurine tracts which are i nterrupted with different b ases in the centre [ 33]. T hey contain t he same sequence in opposite orientations, they were radiolabelled so as to visualize the purine-rich strand of DMG60R, and pyrimidine-rich strand of DMG60Y. AG1 and GA1 contain th e sequen ces A 6 G 6 .C 6 T 6 and G 6 A 6 .T 6 C 6 inserted into the BamHI site of pUC18 [34]. Radiolabelling visualizes the purine-rich strand of AG1, but the p yrimidine-contain- ing s trand o f G A1. F ragments WPseq 1 and WPseq2 were obtained by cloning appropriate oligonucleotides into the BamHI site of pUC18. The sequences were confirmed by manual sequencing with a T7 sequencing kit (Amersham Pharmacia). Fragment W Pseq2 was found to contain a dimer of the required i nsert. These fragments were obtained by cutting the plasmids with HindIII and SacIandtheywere labelled a t the 3¢-end of the Hin dIII site with [ 32 P]dATP[aP] using reverse transcriptase. Radiolabelled DNA was s epar- ated from the remainder of the plasmid on 6–8% non- denaturing polyacrylamide gels. The bands containing the radiolabelled DNA were excised and eluted into 10 m M Tris/HCl pH 7.5, containing 0.1 m M EDTA. The DNA was then precipitated with ethanol in the p resence of 0.3 M sodium acetate. Fig. 1. Chemical structure of WP631. The carbon atoms at positio ns 13, 14, 3 ¢ and 4 ¢ are i ndicated in the upper part of the dimer. Ó FEBS 2004 WP631 sequence selectivity (Eur. J. Biochem. 271) 3557 DNase I footprinting Radiolabelled DNA was dissolved in 10 m M Tris/HCl pH 7.5 c ontaining 0.1 m M EDTA, at a bout 10–20 c.p.s. ÆlL )1 as determined on a hand-held Geiger counter. This DNA solution (1.5 lL) was m ixed with 1.5 lL of ligand (final concentration 10 n M )10 l M ), dissolved in 10 m M Tris/HCl, p H 7 .5, containing 10 m M NaCl. This mixture was allowed to equilibrate for at least 30 min b efore digesting with either DNase I or a hydroxyl radical generating mixture as p reviously described [31]. DNase I digestion was achieved by adding 2 lL enzyme (typically 0.01 UÆmL )1 ) dissolved in 20 m M NaCl, 2 m M MgCl 2 ,and 2m M MnCl 2 . The digestion was stopped a fter 1 min by adding 5 lL o f 80% formamide c ontaining 10 m M EDTA, 10 m M NaOH and 0 .1% (w/v) bromophenol blue. Hydroxyl radical footprinting Hydroxyl radical cleavage was performed by adding 6 lLof a freshly prepared mixture containing 50 l M ferrous ammonium sulf ate, 100 l M EDTA, 2 m M ascorbic acid and 0.05% hydrogen peroxide. The reaction was stopped after 10 m in by precipitating with e thanol. T he D NA w as finally redissolved in 8 lL of 80% formamide containing 10 m M EDTA, 10 m M NaOH and 0.1% (w/v) bromo- phenol blue. Reaction with diethylpyrocarbonate and potassium permanganate The reaction with these footprinting probes was performed as previously described [ 29,31]. Radiolabelled DNA (3 lL) was incubated with 3 lL WP631 diluted t o appropriate concentrations in 10 m M Tris/HCl containin g 10 m M NaCl and equilibrated for at least 30 min. For diethylpyrocarbo- nate (DEPC) modification, 5 lL o f DEPC w as added a nd the reaction was stopped a fter 20 min by precipitating with ethanol in the presence of 0.3 M sodium acetate. For reaction with permanga nate, 1 lLof100m M potassium permanganate was added a nd the reaction s topped after 1 min by adding 2 lL o f mercaptoethanol. T he DNA was then precipitated with ethano l in the presence of 0.3 M sodium acetate. For both DEPC and permanganate the dried DNA pellets were boiled i n 10% (v/v) piperidine f or 30 min, reduced to dryness in a Sp eedvac, and redissolved in 8 lL o f 80% form amide containing 10 m M EDTA, 10 m M NaOH and 0 .1% (w/v) bromophenol blue. Fig. 2. DNase I, DEPC and KMnO 4 foot- prints showing the inte raction of WP631 with tyrT(43–59). WP631 concentrations ( lM) are shownatthetopofeachgellane;ÔconÕ cor- responds to cleavage in the absence of added ligand. Tracks labelled ÔGAÕ are markers specific for purines. 3558 K. R. Fox et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Denaturing gel electrophoresis The products o f footprintin g reactions were resolved on 6–10% polyacrylamide gels (depending on the location of the t arget site) containing 8 M urea. DNA samples were boiled f or 3 min immediately before loading onto the gels. Polyacrylamide gels (40 cm long) were run at 1500 V for 2 h . These we re then fixed in 10% acetic acid, transferred to Whatmann 3M paper, dried under vacuum at 80 °Cand exposed to a phosphorimager screen (K odak) overnight. Dry gels were exposed to a Kodak Phosphor Storage Screen, w hich was s canned using a Molecular Dynamics Storm 860 phosphorimager. The pr oducts of digestion were assigned by comparison with Maxam–Gilbert marker l anes specific for g uanine and adenine. Differential cleavage plots The i ntensity of bands i n the drug-treated and control lanes were prepared as previously described [31]. In the differen- tial cleavage plots the intensity of each band in the drug- treatedlaneisdividedbytheintensityofthesamebandin the drug-free control. These values are then normalized according to the total intensity of the bands in each lane. The v alues are then plotted against the DNA sequence on a logarithmic scale. Values less than one correspond to regions of p rotection by t he ligand, while values greater than one correspond to drug-induced enhanced cleavage. Results Figure 2 shows footprinting gels for the interaction of WP631 with the tyrT DNA fragment. This fragment has been widely used for assessing the s equence-specific bind ing of small m olecules to DNA including the a nthracycline antibiotics daunorubicin and nogalamycin [17,19,35,36]. The first panel shows the results of DNase I f ootprinting, from wh ich it is clear tha t the ligand has af fected the cleavage pattern. At the highest concentrations of WP631 (2 and 3 l M ), the ligand shows a general i nh ibition o f cleavage at most positions in the fragment. However specific regions of protection are evident with ligand c oncentrations between 0.2 and 1 l M . Examples of bands that are protected by the ligand include positions 34, 41, 53 and 61. In contrast, cleavage at positions 31–32 and 47–50 is enhanced in the presence of the ligand. These r esults are presented as a differential cleavage plot in the top panel of F ig. 3 , showing the intensity of each band in the drug-treated lanes compared with that in the control. Examination o f the patterns does not reveal any obvious sequence p reference, though some o f the clearest footprints are located in regions containing both G and A residues. The enhancements are located in oligo(dA) tracts, as often noted with intercalating agents. These footprints are of variable lengths. The footprints around positions 40, 63 and 8 0 c over about six bases, as might be e xpected for a bis-intercalator. However, below position 60 t here are two smaller f ootprints of about Fig. 3. Differential c leavage plots showing the i nteraction of WP631 with tyrT(43–59), AG1 and GA1. The plots were calculated from th e cleavage patterns in the presence of 1 l M WP631 shown in F ig. 2 (tyrT)andFig.6(AG1andGA1).Onlyapartof each sequence is s hown and is written reading 5 ¢)3¢ from left to right; the right-hand e nd corresponds to the bottom of the gels. The ordinate, w hich is plotted on a logarithmic scale, shows the intensity of e ach band i n the drug-treate d lanes relative to that in the control. Values lessthanonecorrespondtoprotectionbytheligand, while values above indicate enhanced cleavage. The black b ars highlight the regions that are p rotected from cleavage. For tyrT t he arrows indicate the positions of WP631-induced c leavage by DEPC (grey arrows) and KMnO 4 (black arrows). Ó FEBS 2004 WP631 sequence selectivity (Eur. J. Biochem. 271) 3559 three base pairs each. Similar s hort footprints are apparent around positions 25 and 34. As DNa se I overestimates ligand binding site sizes by 3–4 base pairs i t is very unusual to observe footprints of this short size. It is possible that these short f ootprints are no t caused by steric interference from drug molecules bound to the DNA minor groove, instead they may r eflect drug-induced changes in DNA structure that render it less sensitive to cleavage. We attempted to gain more a ccurate information about the sequence specificity of WP631 by performing hydroxyl radical footprinting experiments . However t he ligand d id not affect hydroxyl radical cleavage at a concentration of 10 l M . A lthough this result is disappointing, some other well-characterized sequence specific ligands also fail to produce hydroxyl r adical footprints [36,37]. Because o f the difficulty in interpretin g these patterns, we examined the effect of WP631 on modification by DEPC and potassium permanganate. These agents react with exposed A and T residues, respectively, while duplex DNA is generally u nreactive [31]. Intercalating age nts have previously been shown to enhance the reactivity of bases adjacent to their binding sites to t hese agents [ 38–40]. T he results a re presented in the second and t hird panels of Fig. 2. It can b e s een that WP631 enhances the r eactivity o f certain bases to each of these agents; these are indicated by the arrows in Fig. 3. Bands that become hyper-reactive to DEPC are located at positions 18, 32, 48, 67, 83 and 84. In some instances these a re located i n regions o f enhanced DNase I cleavage (positions 32 and 48), while others are adjacent to regions of DNa se I protection (18, 67, 83, 84) . Enhanced reactivity to KMnO 4 can be seen at posit ions 29, 33, 60, 6 8, 81, 86, 88 and 9 1. These results show that WP631 produces distinct foot- printing patterns, w hich are different to those produced by daunorubicin and nogalamycin [17,19,35,36]. The ligand must therefore possess some sequence selectivity, though no consensus b inding sites can be deduced from these patterns. We have therefore examined the interaction of this ligand with a r ange of DN A fragments, i n o rder to elucidate the characteristics of t he preferred binding sites. Fig. 4. DNase I and KMn O 4 footprints showing the interaction of WP631 with fragments MS1 and MS2. WP631 concentrations (l M )areshownat the top of each gel lane; ÔconÕ corre sponds to cleavage in the absence of added liga nd. Tracks labelled ÔGAÕ are markers specific for purines. The numbered black bars s how the positions of D Nase I footprints, while the aste risks indicate b ands that be come sensitive t o reaction with K MnO 4 in the presence of W P631. 3560 K. R. Fox et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fragments M S1 and MS2 were designed so as to contain all 136 tetranucleotide sequences [32]. They contain i dentical sequences, but are cloned i n opposite orientations thereb y simplifying analysis of bands at the ends of the fragments. Footprinting exper iments w ith these fragments are p resen- ted in F ig. 4 and differential c leavage plots derived from these d ata are shown in Fig. 5 . Again it is clear t hat WP631 has altered the cleavage patterns, producing footprints that are highlighted by the bars in F igs 4 and 5. Several of these footprints are l ocated in regions which are rich in GA (TC) or GT (AC) residues, for example sites 1 (TCATCTC), 2 (GGTGG), 4 (GAAGAG), 7(ATGTGT), and 8 (GTTGG). A long footprint is also evident on MS2 (site 10) corresponding to a purine-rich tract. These footprints are accompanied by enhancements in reac tivity to KMnO 4 andDEPCasindicatedinFigs4and5. As many of the footprints on MS1 and MS2 are located in tracts of GA-residues we examined t he interaction w ith other fragments containing similar sites, some o f which were prepared for work with triplex-forming oligonucleotides. Fragments G A1 and AG1 contain t racts of G 6 A 6 .T 6 C 6 and A 6 G 6 .C 6 T 6 , r espectively [34]. DNase I footprinting patterns for WP631 with these fragments are shown in Fig. 6 and differential clea vage plots derived from these are present ed in Fig. 3. As these oligop urine tracts were both cloned into the polylinker s ite o f p UC18 the sequences surrounding the inserts are common to both fragments and show similar cleavage patterns in the presence of the ligand. For both fragments there is a large footprint below the insert, corresponding to the sequence TCCTCT. Similarly cleavage is attenuated above the inserts in vicinity of the sequence GGATC. H owever the ligand h as very different effects on cleavage of the two inserts. WP631 protects from D Nase I cleavage at the centre of AG1, but causes enhanced cleavage at the centre of GA1. It therefore appears that A n G n is a much better binding site than G n A n . Fragment DMG60 a lso c ontains oligopurine tracts that are interrupted by isolated thymine r esidues [ 33]. DNase I digestion p atterns for the pyrimidine-rich strand of this fragment i n the presence of WP631 are shown in Fig. 6 and differential cleavage plots for both strands are shown i n t he bottom panel of Fig. 5. Two clear footprints can be seen on this fragment (labelled sites 1 a nd 2), as well as o ther regions of protection a t t he top and bottom of t he gel, which are in the remainder of the polylinker. A short region of protection is also evident around the l owest purine r esidue (arrowed). The strong footprints correspond to sequences TTCTTC (site 1) and TTTCTTT (site 2). Although these both contain the sequence TTCTT, this alone cannot constitute the preferred ligand binding site as the same pentanucleotide is present in other positions which are not protected. These will be considered further in the Discussion. As several of the footprints identified a bove are located in GA (CT) or GT (AC) tracts w e prepared a new fragment (WPseq2) containing five variations on the h exanucleotide sequence SWSWWS (S ¼ GorC,W¼ A or T), in which the different s ites are separated b y C C (GG). T he results o f DNase I footprinting experiments with this fragment a re Fig. 5. Differential cleavage pl ots showing the interaction of WP631 with MS1, MS2 and DMG60. The plots were calculated from the c leavage patterns i n the prese nce of 1 l M WP631 s hown in Fig. 4 (MS1 and MS2) and Fig. 6 (DMG 60Y). Only a part of each sequence is shown and is written reading 5¢)3¢ from left to right; the r ight-han d end corresponds t o the bottom of t he gels. The ordin ate, which is plot ted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control. Values of less than one correspond t o protection by the ligand, while values above indicate enhanced cleavage. The black bars highlight the regions th at are protected from cleavage. F or MS1 and MS2 the arrows indicate the p ositions of WP631-induced cleavage by DEPC (grey arrows) and KM nO 4 (black arrows). Ó FEBS 2004 WP631 sequence selectivity (Eur. J. Biochem. 271) 3561 shown in Fig. 7 . It can be seen that there a re footprints at all the potential sites, which a re most clearly seen in the differential cleavage plot. T he strongest s ites are a t GTGTTG and CTTCTC. There is little or no protection in the junctions between t he various sites a nd there i s enhanced cleavage between GTGGTG and CCACAC. These r egions of protection a re located towards the 3¢-end of each target site as normally observed w ith D Nase I footprinting, as th is enzyme c uts across t he width of the DNA minor groove. Daunorubicin is thought to bind best to sequences of the type 5¢-(A/T)CG and 5¢-(A/T)GC [17–19] and previous NMR and crystallographic studies with bis-daunorubicins have investigated their interaction with CGTACG and TGTACA [28,29]. None of these sequences are r epresented in any o f the footprinting substrates mentioned a bove. We therefore prepared a novel fragment (WPseq1) containing the sites CGATCG, CGTACG, GCATGC, GCTAGC and TGTACA each separated b y the sequence A ATT t o which the drug is not expected to bind. The r esults of footprinting experiments with t his fragment a re presented i n Fig. 8 . T he DNase I c leavage patterns show footprints at each of these sites, some of which persist to between 0.1 a nd 0.2 l M .The positions of these sites are confirmed i n the differential cleavage plot shown i n Fig. 8(B). Although DNase I footprinting cannot usually be used to determine ligand binding sites t o single b ase r esolution, some interesting features of WP631 binding can be deduced by comparing the protection at each of these potential sites. The centr al portions of the differential cleavage plots a re four bas es long for GCATGC, CGATCG and TGTACA and each begin at the second base. These footprints are symmetrically located around the centre o f e ach h exanucleotide t arget, whereas DNase I footprints are us ually staggered t owards the 3¢-end. In contrast the f ootprint at GCTAGC is l onger and begins one base before the s tart of this hexanucleotide; it is th erefore staggered to wards t he 5 ¢-end of the h exa- nucleotide site. The footprint at CGTACG appears to consist of two smaller r egions and there is little p rotection at the central adenine. I t should be r emembered that a ll these sites are symmetrical (palindromic) s equences. If the ligand binds to one side of the site then a second identical site will be present i n t he other half of t he hexanucleotide ( i.e. i f i t binds to GCTAGC by recognizing GCT, t hen a second identical binding site must be present in the other h alf of t he sequence at A GC). Although t wo ligand m olecules will not be able to bind simultaneously, the average of the t wo equivalent binding sites would be a larger footprint, which is not wh at we observe. It t herefore seems most likely that a single ligand molecule is bound across the centre of each site. T hese differences between these sites are also evident in the patterns o f DEPC e nhancement, which are indicated by the arrows in Fig. 8. There i s e nhanced DEPC reactivity at the first adenine after the hexanucleotide s ite (AATT) for Fig. 6. DNase I footprints s howing the inter- action of WP631 with f ragments AG1, GA1 and DMG60Y. WP631 concentrations (l M ) are shown at the t op of each gel lane; ÔconÕ corresponds to cle avage in the absence of added ligand. Tracks labelled ÔGAÕ are m ark - ers s pe cific for purines. The nu mbered black bars show the positions of DNase I foo tprints with DMG60Y. 3562 K. R. Fox et al. (Eur. J. Biochem. 271) Ó FEBS 2004 GCATGC and GCTAGC, while this is at the second adenine (AATT) for CGATCG and TGTACA. T here is no enhancement i n r eactivity t o DEPC after CGTACG. These subtle differences suggest that WP631 does not have exactly the same m ode of binding at each of these s ites. Discussion The footprinting results p resented in this paper demonstrate that WP631 binds to DNA in a sequence s elective fashion and that i ts preferred binding sites a re different from t hose of daunorubicin and nogalamycin. By c omparison with daunorubicin it was expected that WP631 should bind best to sequences such as CGATCG and CGTACG, which have been used in X-ray and NMR structural studies with this ligand [28,29]. The experiments with fragment WPseq2 confirm that WP631 does indeed bind to this site at concentrations as low as 0 .2 l M , but experiments with t his and other fragments show that it also b inds equally well to other sequences. Another difference between these patterns a nd those produced by daunorubicin is t heir temperature d ependence. Previous studies with daunorubicin [17–19] only detected DNase I f ootprints at low t emperature (4 °C) presumably as this slows the dis sociation of the ligand f rom DNA; no footprints were ob served at 20 °C. In contrast WP631 produces clear footprints at 20 °C which are still apparent at 37 °C. In this case we observe no WP631 footprints at 4 °C. This could be because the DNA becomes too rigid to permit bis-intercalation, or because s elf-stacking of the ligand i s favoured at lower temperatures. Mode of binding Although the present work does not directly concern the mode of binding of WP631, this will influence the interpretation of the footprinting patterns. Previous struc- tural work has demonstrated that WP631 binds in the minor groove of CGTACG with four base pairs sandwiched between the intercalating chromophores. In c ontrast, t he related compound WP652, in which t he dimer is connected via C4¢, binds to the Y GTR steps in TGTACA, sandwich- ing o nly two base pairs between the chromophores. The precise orientation of the xylyl gr oup of WP631 is also different in the two structures in which it is either perpendicular o r parallel to the walls of the minor groove. Fig. 7. Interaction o f WP631 with fragment W Pseq1 . (A) DNase I f ootprints showing the interaction of WP631 with fragment WPseq2. WP631 concentration s (l M )areshownatthetopofeachgellane;ÔconÕ corresponds to cleavage in the a bse nce of added ligand. Trac ks labelled ÔGAÕ are markers specific for purines. The potential hexanucleotide binding sequences are indicated alongside the gel. (B) Differential cleavage plot showing the interaction of WP631 with WPseq2. The plots were calculated from the cleavage patterns in the presence of 1 l M WP631 shown in Fig. 7A. Onlyapartofeachsequenceisshownandiswrittenreading5¢)3¢ fro m left to right; the r ight-hand end co rrespo nds to the b ottom o f the gel. The ordinate, which is plotted on a logarithmic s cale, shows t he intensity of eachbandinthedrug-treatedlanesrelative t o that in the c ontrol. Values less thanonecorrespondtoprotectionbytheligand,while values above indicate enhanced cleavage. The vertical lines divide the f ragment into t he various h exanucleo tide repeats. Ó FEBS 2004 WP631 sequence selectivity (Eur. J. Biochem. 271) 3563 These different str uctures suggest that WP631 may b ind to different sequences in different modes, sandwiching between two a nd four base pairs b etween the c hromophores. These different modes will depend on the local DNA structure and flexibility as well a s any contacts between the ligand and its binding site. A further complication is the possibility that WP631 might bind to some sequences by mono-intercala- tion, leaving the second chromophore i n free solution or stacked within t he groove. The possibility of additional sequence-specific groove binding may further complicate the footprinting pattern. The coexistence of different binding modes is suggested by the footprinting d ata presented i n t his paper. Some binding sites are six to eight base pairs long, as expected for a ligand that spans six base pairs, while others are much shorter, and appear to cover only t hree bases . Thes e r esults are consistent with a recent study sugges ting that WP631 can bind in two d ifferent modes with stoichiometries of 6 : 1 a nd 3 : 1 base pairs per drug [41]. Sequence selectivity The results with these DNA fragments show that WP631binds t o DNA in a sequence s elective fashion, as specific footprints are g enerated at moderate ligand c on- centrations (about 0.3 l M ). At high concentrations (3 l M and above) the ligand is able to bind t o most s ites, as s hown by the general inhibition of DNase I c leavage. Examination of the footprints does not reveal the p resence o f a ny particular di- or tri-nucleotide step within the binding sites, though many of the protected regions are G A or GT-rich in one strand, and there ar e no f ootprints in GC- or AT-rich sequences. The results with MS1 and MS2, which contain every possible tetranucleotide combination, d emonstrate that WP631 d oes not bind to a unique tetranucleotide, though we cannot exclude the possibility that it binds especially well to a unique hexanucleotide w hich is not represented in these fragments. S everal of the footprints are found in oligopurine-oligopyrimidine sequences, especially those seen with fragment DMG60. In the published crystal [29] and NMR structures [28], WP631 is bound to the sequences CGATCG and CGTACG, with the chromo- phores intercalated between each o f the CpG s teps. This sequence is present in fragment W Pseq2 and is i ndeed part of a clear DNase I footprint, tho ugh several other sequences produce equally good footprints on this fragment. It i s therefore clear that W P631 can bind to m any s ites w ith the general sequence (G/C)(G/C)(A/T)(A/T)(G/C)(G/C). A footprint i s also evident in this fragment at the sequence TGTACA, which was s uggested as one of the potential binding sites for WP652 [29]. We therefore examined the footprinting results on all the fragments for degenerate sequences th at might form t he preferred binding sites. We find that footprints are often foun d around the sequence (G/C)(A/T)(A/T)(G/C), and that there a re no occasions when this is not part of a drug b inding site. For example, on Fig. 8. Interaction of WP631 with fragment WPseq2. (A) DNase I and DEPC foo tprints. W P631 c oncentrat ions (l M )areshownatthetopofeach gel lane; ÔconÕ co rresponds to cleavage in the absence of added ligand. Tracks labelled ÔGAÕ are m arkers specific for purines. The p otential hexanucleotide binding sequences are indicated alongside the gel. (B) Differential cleavage plot s howing the interaction of WP631 with WPseq1. The plot wa s calculated from the cleavage patterns in the pre sence of 0.2 l M WP631 shown i n Fig. 8A. Only a part o f each s equence is s how n and is written r eading 5¢)3¢ from l eft to righ t; the r ight-hand end corresponds to the bottom o f the gel. The o rdinate, which is plotted on a logarithmic scale, shows the intensity of each b and in the drug -treated lanes relative to that in the control. Valuesoflessthanonecorrespond to protection by the ligand, while values above one indicate enhanced c leavage. The arrows indicate the positions of W P631-induced cleavage by DEPC. 3564 K. R. Fox et al. (Eur. J. Biochem. 271) Ó FEBS 2004 MS1 the foot prints are at s ite 1 (CATC), s ite 3 (GTAC) and site 4 (GAAG), while on MS2 they are seen at site 7 (CATG), s ite 8 (GTTG), s ite 9 (CTTG and GATC). In addition the weaker regions of protection between sites 8 and 9 contain the sequences CTAC and CTAG. This consensus sequence is also found on the tyrT fragment at positions 25 (CATC), 38 (GTTG), 43 (GAAC) and 5 7 (GAAG) each of wh ich corresponds to a r egion t hat is protected by t h e ligand. The s equences GATC and C TGA are a lso f ound in the polylinker r egions of pAG1 and pGA1, and at site 2 in DMG60 (CTTC). We therefore suggest that WP631 binds well to the sequence (G/C)(A/ T)(A/T)(G/C). However, t his s equence c annot be the only good ligand b inding site. F or example, the f ootprint at t he centre of pAG1 contains the sequence AGGG (in contrast to GGGA, which does not produce a footprint with pAG2). Moreover sites 2, 5 and 6 on MS1 are found around the sequences GGTG ( or GTGG) (site 2), TTAG (site 5) and GTATAG (site 6). The footprint at s ite 2 of DMG60 also does not fit this pattern, and at this position it seems likely thattheligandisabletobindtothetwoadjacentsites CTTT. It therefore appears that the ligand can also bind well to some sites in w hich one or more base does not match the predicted pattern. It should be noted that, although t hese results show the presence of specific binding sites f or WP631, these a re typically only evident at concentrations of 0.3 l M and above. In contrast, previous studies have suggested that WP631 binds with an association c onstant of 3 · 10 11 M )1 [27], from which we would expect footprints to persist to much lower (subnanomolar) concentrations. A n umber of factors may contribute to this d ifference. First, i t i s possible that the preferred b inding site is not represented i n the footprinting s ubstrates that we have used. 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Kutsch, O., Levy, D.N., Bates, P.J., Becker, J., Kosloff, B.R., Shaw, G.M., Priebe, W. & Benveniste, E.N. (2004) Bis- Ó FEBS 2004 WP631 sequence selectivity (Eur. J. Biochem. 271) 3565 [...]... investigated by DNase I footprinting Biochemistry 25, 4349–4356 36 Fox, K.R (1988) Footprinting studies on the interactions of nogalamycin, arugomycin, decilorubicin and viriplanin with DNA Anti-Cancer Drug Design 3, 157–168 37 Churchill, M.E.A., Hayes, J.J & Tullius, T.D (1990) Detection of drug binding to DNA by hydroxyl radical footprinting: relationship of distamycin binding sites to DNA structure... Chaires, J.B (1998) Ultratight DNA binding of a new bisintercalating anthracycline antibiotic Biochemistry 37, 1743–1753 Robinson, H., Priebe, W., Chaires, J.B & Wang, A.H.J (1997) Binding of two novel bisdaunorubicins to DNA studied by NMR spectroscopy Biochemistry 36, 8663–8670 Hu, G.G., Shui, X.Q., Leng, F.F., Priebe, W., Chaires, J.B & Williams, L.D (1997) Structure of a DNA bisdaunomycin complex Biochemistry... Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand Biochemistry 39, 6714–6725 Stonehouse, T.J & Fox, K.R (1994) DNase I footprinting of triple helix formation at polypurine tracts by acridine-linked oligopyrimidine Biochim Biophys Acta 1218, 322–330 Ó FEBS 2004 35 Fox, K.R & Waring, M.J (1986) Nucleotide sequence binding preferences of. .. Vaquero, A., Ferrer, N., Villamarin, S & Priebe, W (2001) Analysis of the effects of daunomycin and WP631 on transcription Curr Med Chem 8, 1–8 Fox, K.R & Waring, M.J (2001) High resolution footprinting studies of drug DNA complexes using chemical and enzymic probes Methods Enzymol 340, 412–430 Lavesa, M & Fox, K.R (2001) Preferred binding sites for [N-MeCys3,N-MeCys7]TANDEM determined using a universal... positioned nuclesomes on 5S RNA genes of Xenopus Biochemistry 29, 6043– 6050 38 Portugal, J., Fox, K.R., McLean, M.J., Richenberg, J.L & Waring, M.J (1988) Diethyl pyrocarbonate can detect a modified DNA structure induced by the binding of quinoxaline antibiotics Nucleic Acids Res 16, 3655–3670 39 Jeppesen, C & Nielsen, P.E (1988) Detection of intercalationinduced changes in DNA structure by reaction with... structure by reaction with diethylpyrocarbonate or potassium permanganate Evidence against the induction of Hoogsteen base pairing by echinomycin FEBS Lett 231, 172–176 40 Fox, K.R & Grigg, G.W (1988) Diethylpyrocarbonate and permanganate provide evidence for an unusual DNA conformation induced by binding of the antitumour antibiotics bleomycin and phleomycin Nucleic Acids Res 16, 2063–2075 41 Haj, H.T.-B.,... phleomycin Nucleic Acids Res 16, 2063–2075 41 Haj, H.T.-B., Salerno, M., Priebe, W., Kozlowski, H & GarnierSuillerot, A (2003) New findings in the study on the intercalation of bisdaunorubicin and its monomeric analogues with naked and nucleus DNA Chem Biol Interacts 145, 349–358 . examine the binding of WP631 to DNA [27]. Continuous variation Correspondence to K. R. Fox, Schoo l of B iological Sciences, U niver- sity of Southampton, Bassett. range of natural a nd synthetic footprinting substrates to examine t he sequence- selective i nteraction of the bis-daunorubicin anti- biotic WP631 with DNA.