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..) 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 24 6725-6730 Echinomycin binding to alternating AT Keith R.Fox, Jonathan N.Marks and Kerstin Waterloh Department of Physiology & Pharmacology, University of Southampton, Bassett Crescent East, Southampton S09 3TU, UK Received October 18, 1991; Revised and Accepted November 21, 1991 ABSTRACT We have studied the binding of echinomycin to DNA fragments containing GC-rich regions flanked by blocks of alternating AT by DNase I footprinting and diethylpyrocarbonate modification. Regions of alternating AT flanking the sequences CCCG, CCGC, CGGC and GG show a four base pair DNase I cleavage pattern and reaction of alternate adenines with diethylpyrocarbonate. This pattern is strongest when the AT-block is immediately adjacent to the CpG ligand binding site. We explain these phenomena by suggesting that echinomycin binds to the dinucleotide step ApT in a cooperative fashion. The cooperative effects can be transmitted through the dinucleotide step GC but not CC or AA. No such repetitive patterns are seen with surrounding regions of (ATT).(AAT). Evidence is presented for secondary drug binding sites at CpC and TpG with weaker interaction at the CpG site within the hexanucleotide TTCGAA. INTRODUCTION The bifunctional intercalator echinomycin has long been known to bind to GC-rich DNAs [1]. More recently footprinting, NMR and crystallographic studies [2-6] have demonstrated that the ligand is selective for the dinucleotide step CpG. However a paradox remains since although its binding constants to natural DNAs correlate with their gross G+C content, the drug binds appreciably well to poly(dA-dT) with K(0) =0.3 x 106M- I as compared with 0.55 x 106M-' for poly(dG-dC) [1]. This discrepancy is even greater for the related antibiotic triostin A which binds to poly(dA-dT) better than poly(dG-dC) [7]. Despite this observation both ligands do not yield footprints at ApT or TpA. The interaction with poly(dA-dT) is highly cooperative, possibly explaining the inability to footprint the drugs on shorter AT-rich sequences. Alternatively the absence of a footprint could be a kinetic phenomenon since echinomycin has been shown to dissociate from poly(dA-dT) faster than from poly(dG-dC) [8]. Recent footprinting studies using DNA fragments containing regions of alternating AT adjacent to CpG sites revealed that echinomycin caused alternate adenines to become hyperreactive to diethylpyrocarbonate [9] and induced an unusual four base pair repeat pattern in DNase I digests [10]. In this paper we have examined the effects of echinomycin on DNase I digestion and diethylpyrocarbonate modification of a series of DNA fragments containing regions of alternating AT positioned at various distances from CpG sites. MATERIALS AND METHODS Drugs and enzymes Echinomycin was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. It was stored as a 1mM stock solution in dimethylsulphoxide and diluted to working concentrations immediately before use. DNase I was purchased from Sigma and stored as previously described [2]. The oligonucleotides (AT)1OSSSS(AT)1O, (S=G or C), (AT)15SS(AT)15, (ATT)4CG(AAT)4 and (TAA)4CG(TTA)4 were prepared on an Applied Biosystems DNA synthesizer and used without further purification. DNA fragments The self complementary oligonucleotides were cloned into the SmaI site (CCC/GGG) of pUC 19 and transformed into E. coli TG2 as previously described [ 1]. The sequences were confirmed by dideoxy sequencing with a T7 polymerase kit (Pharmacia). Each of the plasmids was found to contain a single copy of the synthetic insert except (TAA)4CG(TTA)4 which contained a dimer. Three different clones were characterised from oligonucleotide (AT)1OSSSS(AT)1O containing central tetranucleotides CCCG, CCGC and CGGC. A plasmid containing the insert (AT)15GG(AT)6 was obtained from the oligonucleotide (AT)15SS(AT)15. PUC13 plasmids containing the inserts (AT)5, T(AT)6 and (AT)10 were prepared as previously described [12]. Plasmids were purified using Qiagen columns according to the manufacturers instructions. Radiolabelled DNA fragments containing the synthetic inserts were prepared by digesting with HindIll, labelling at the 3'-end with [a-32P]dATP using reverse transcriptase and cutting again with either EcoRl or KpnI. The labelled fragments of interest (about 75 base pairs) were separated on 7 % polyacrylamide gels. Footprinting and gel electrophoresis Footprinting experiments with DNase I and diethylpyrocarbonate were performed as previously described [2,9 - 14]. The products of digestion were resolved on denaturing polyacrylamide gels (8% for fragments labelled at the HindHI end, 12% for fragments labelled at the EcoRl end) containing 8M urea. These were then 6726 Nucleic Acids Research, Vol. 19, No. 24 fixed in 10% (v/v) acetic acid, transferred to Whatman 3MM paper, dried under vacuum at 80°C, and subjected to autoradiography at -70°C with an intensifying screen. Autoradiographs were scanned with a Joyce-Loebl Chromoscan 3 microdensitometer. RESULTS (AT). regions surrounding CpG sites Figure 1 presents DNase I digestion patterns in the presence and absence of echinomycin for DNA fragments containing the inserts (AT) IOCCCG(AT)lo, (AT)IOCCGC(AT),O and (AT)1OCGGC (AT)1O (henceforth known as CCCG, CCGC and CGGC respectively). In each case cleavage in the control consists of an alternating pattern of bands in which cleavage of ApT is much better than TpA; products from the latter are barely visible. This alternation is interrupted around the four central CG base pairs. In each case a clear footprint is formed in the central GC-rich region. The position of the CpG step is different for the three fragments; protections extend into the second ApT step on the 3'-(lower) side for CCCG, but only the first ApT for CCGC and CGGC. Protection to the 5'-(upper) side of the CG follows the same trend; no ApT bonds on the 5'-side of CCCG and CCGC are protected; though when the drug binding site is shifted by one more base in the 5'-direction, in CGGC, the first ApT bond is protected. As well as producing footprints around the central GC-rich region echinomycin induces unusual cleavage patterns in the surrounding blocks of alternating AT, dramatically increasing the cleavage of some, but not all, the intervening TpA steps. These results are qualitatively similar to those reported in the preceding paper for T(AT)8CG(AT),5 [10]. This can be seen more clearly in the cleavage histograms presented in Figure 2. Looking first at the results for CCCG we see a distinct four base pair repeat pattern on the 3'-side in which every other TpA step is cut well, the other TpA steps remain poorly cleaved as in the control. Alternate ApT bonds are also cut with different efficiencies. On the 5'-side, further from the drug binding site, the pattern is more even with each TpA step enhanced to a similar extent. The unusual four base pair repeat pattern is again evident with CCGC. nTe pattern on the 3'-side is out of phase with that seen with CCCG. With CCCG the 4th, 6th and 8th ApT steps are cut best, whereas for CCGC it is the 3rd, 5th, 7th and 9th. With CCGC a four base pair pattern is also evident on the 5'-side. This is most clearly seen as an alternating cleavage pattern of adjacent TpA bonds; the uneven cleavage pattern of ApT fades out towards the end of the fragment. A similar pattem is evident on both sides of CGGC, though the first two bonds on the 3'-side do not fit with the rest of the fourfold repeat. The fourfold pattern on the 3'-side is in phase with CCGC, but out of phase with CCCG. Figure 3 presents the results of diethylpyrocarbonate modification of these DNA fragments in the presence of A :::!" .-"':': ILhLJI :,:;f 4;--t 'I.: 7- ::---- 1, ,,I. .I... ILftU ATATATATATATATATATATCCCGATATATATATATATATATAT .: -.1.1. B I I 0 1 11 I I IiI.IiI.II ATATATA TAT A T AT ATAT ATCCGCATATATATATATATATATA T C .1 .1 .1i.1,I Figure 1. DNase I digestion pattern of DNA fragments containing the inserts (AT)1OCCCG(AT)IO, (AT)IOCCGC(AT)1O and (AT)1OCGGC(AT)1O in the presence and absence of echinomycin. The square brackets indicate the position and length of the inserts. Arrows indicate the position of the CpG sites. Each pair of lanes corresponds to digestion by the enzyme for 1 and 5 minutes. Drug concentrations (juM) are shown at the top of each pair of lanes. The tracks labelled 'G' are dimethylsulphate-piperidine markers specific for guanine. CCCG is a HindIII-EcoRl fragment while CCGC and CGGC were obtained by digesting with HindHI and Kpnl. All DNA fragments were labelled at the 3'-end of the HindIII site. ATATATATATATA TATATATCGGCATATATATATATATATATAT Figure 2. DNase I cleavage histograms for the fragments containing the inserts (AT),OCCCG(AT)IO (A), (AT)IOCCGC(AT)IO (B) and (AT)1OCGGC(AT)IO (C) in the presence of IOOtM echinomycin. Sequences are written in the usual convention i.e. 5' to 3', reading from left to right. The bars indicate the relative cleavage at each bond determined from densitometer scans of the data presented in Figure 1. Only regions outside the footprint have been included. Nucleic Acids Research, Vol. 19, No. 24 6727 echinomycin. This probe interacts only weakly with native BDNA and produces virtually no cleavage in the control. Echinomycin has increased the reactivity of adenines throughout each of the inserts. Looking first at the CCCG fragment the first adenine on the 3'-side of the CG step is strongly enhanced. This is followed by an alternating pattern of enhanced cleavage products so that the first, third and fifth adenines are strongly enhanced. This alternating pattern fades out towards the 3'-end of the insert. Adenines on the 5'-side of the CCCG are all modified with a similar efficiency. DEPC modification of CGGC shows a similar pattern of cleavage at alternate adenines to both side of the canonical drug binding site. This pattern of alternation also fades out towards the ends of the insert. The strongest bands correspond to modification of the 2nd, 4th, 6th and 8th adenines on the 3'-side of fragment and the 1st, 3rd, 5th and 7th adenines on the 5'-side. Similar experiments with CCGC reveal the same trend as seen with CGGC, although the alternation is less pronounced. The strongest bands for CCCG are out of phase with CCGC and CGGC as seen with the DNase I results. In every case the adenines which are most strongly modified by DEPC correspond to ApT steps which are cut less well by DNase I. (AT). surrounding other GC-sites The simplest explanation for these changes in sensitivity to DNase I and DEPC is that the echinomycin has bound within the (AT)n stretches, but that their dissociation kinetics are too fast to yield DNase I footprints. Echinomycin molecules are held in phase by strong binding to the central CpG site, possibly by some cooperative interaction. This will be considered further in the Discussion. To check the effect of the central CpG step on the binding to (AT)n we have performed similar footprinting experiments on DNA fragments containing central GG steps. Figure 4 presents DNase I digestion and DEPC modification of a fragment containing the sequence (AT)15GG(AT)6 in the presence of echinomycin. Although no footprints are observed a fourfold repeat pattern for DNase I cleavage and an alternating pattern of DEPC modification are again evident. Does this mean that these patterns are always produced for echinomycin interacting with (AT)n, irrespective of the presence of a central CpG? Not necessarily since CC (GG) has also been suggested as a secondary echinomycin binding site. This too could force drug molecules to bind in phase, though only if binding to GG is stronger than that to AT. This is confirmed by experiments with fragments containing regions of (TA)n cloned into the same site (not shown). In each case echinomycin caused increased cleavage of all the TpA steps, but no fourfold repeat pattern is evident. In this instance the flanking sequences (CCC/GGGG) represent two or three overlapping secondary (GpG) echinomycin binding sites. As a result they can not force a unique orientation of echinomycin molecules throughout the (AT)n blocks. Binding within the (AT)n stretches is therefore be random and yield an even enhancement at each TpA step. (ATT)n and (TAA)n surrounding CpG sites In order to investigate whether these changes are a general property of AT-rich regions surrounding echinomycin binding sites or are restricted to blocks of alternating AT we have A B A TAT ATAT ATAT ATA TATAT GGA TAT ATA TAT AT A TA T AT ATATATAT ATATATCCGCATATAT A T A TA T A T ATATA T ii . .I=i ATATATATATATATATATATCGGCATATATATATATATATATAT Figure 3. Patterns of diethylpyrocarbonate modification of the fragments containing the inserts (AT)IOCCCG(AT)IO (A), (AT)IOCCGC(AT)IO (B) and (AT)1OCGGC (AT)1O (C) in the presence of 1OOyM echinomycin. The bars indicate the relative cleavage at each bond determined from densitometer scans of the data. I1111111 111H III I A TATAT ATATA TATATATATGGA TA TAT AT A TATT Figure 4. Patterns of DNase I digestion (A) and diethylpyrocarbonate modification (B) for the fragment containing the insert (AT)15GG(AT)6 in the presence of 1OOiAM echinomycin. The bars indicate the relative cleavage of each bond. The first five AT steps are omitted from the 5'-end. 6728 Nucleic Acids Research, Vol. 19, No. 24 performed similar experiments on DNA fragments containing the inserts (TAA)4CG(TTA)4 and (ATT)4CG(AAT)4. Figure 5A,B displays the results for the dimeric insert (TAA)4CG(TTA)4 in the presence and absence of echinomycin. DNase I digestion of the (TTA) regions in the control gives a pattern in which the TpT bond is cut best followed by weaker cleavage of TpA and poor cleavage of ApT (TpT > TpA > ApT). In the (TAA) region the order is ApT > ApA > TpA. In the presence of echinomycin clear footprints are visible around both CpG sites, extending over 6-8 base pairs. The triplet pattern in the rest of the insert is retained though products from the weaker steps (TpA and ApT in the upper and lower halves respectively) are now clearly visible. DEPC modification of this fragment shows weak cleavage of the central adenine in the TAA sequence with no reaction at TAA or TTA. Echinomycin renders the upper adenine of the TAA sequence more susceptible to DEPC modification and induces weak modification at TAA and TTA, the latter is weaker than the former. Each of the TAA and ATT repeats is affected to the same extent. There is no evidence of the fourfold pattern seen with alternating AT. Figure 5C,D presents the results DNase I digestion and DEPC modification the fragment containing the insert (ATT)4CG(AAT)4. DNase I digestion in the control produces a cleavage in which TpT > TpA > ApT for the (ATT) region and ApT > ApA > TpA for the (AAT). In both halves the products of the weakest cleavage reaction are barely visible. In the presence of echinomycin there is no clear footprint around the TCGA site, though bands to either side show an enhanced rate of cleavage. M. :,- X .:;, " ii.16 I V. a4,4 6- Figure 5. DNase I (A,C) and diethylpyrocarbonate (B,D) digestion patterns for fragments containing the dimeric insert of (TAA)4CG(AAT)4 (A,B) and (ATT)4CG(AAT)4 in the presence and absence of echinomycin. Each pair of and lanes in the DNase I digests corresponds to digestion by the enzyme for 5 minutes. Drug concentrations (WL) are shown at the top of the lanes. Square brackets show the position and length of the inserts. Arrows show the position of the CpG sites. This is similar to the effect of echinomycin on T15CGA15 which contains the same central hexanucleotide [10]. Although the relative cleavage pattern remains unchanged products from the weakest steps are now evident. No fourfold digestion pattern is induced. DEPC modification of this fragment shows little cleavage in the drug-free control; only the upper adenine of AAT is cut. In the presence of echinomycin there is a dramatic increase in the susceptibility of the adenine distal to the drug binding site (CGA). In addition echinomycin increases DEPC modification at both adenines of the AAT portion to a similar extent, but has little effect on the upper ATT sequence. DISCUSSION Interaction at (AT)n The results presented in this paper provide compelling evidence for some interaction between echinomycin and (AT)n. In the preceding paper we suggested that the unusual patterns result from secondary binding of echinomycin to the dinucleotide step ApT. This binding is too weak (rapid dissociation?) to yield a footprint but produces DNA structural changes which are sufficiently longlived to be detected by DNase I and DEPC. The patterns observed correspond to the 'shadow' left after the drug has dissociated. Although specific secondary binding to ApT (rather than TpA) has not been rigorously proven we will argue that this is sufficient to explain these results. In order to explain the results we will make several assumptions. Firstly that binding to AT regions adjacent to the CG binding site is highly cooperative and promoted by binding of the ligand to the primary site. Secondly that the neighbour exclusion principle still holds, i.e. that at least two base pairs must separate adjacent drug molecules. Thirdly we assume that the cooperative effects rapidly fade as we move away from the first drug molecule, i.e binding of the second molecule is strongest with two intervening bases, weaker with three bases and effectively non-cooperative with four bases. Fourthly we assume that hyperreactive adenines are found on the 3'-side of the sandwiched base pairs i.e at ATA. Using this model we will first consider the results for DEPC modification of CCCG, CCGC and CGGC. For CCCG the CpG step will be occupied by the ligand under all conditions and result in strong reaction at the first adenine (CGA) on the 3'-side. The neighbour exclusion principle will forbid drug binding at the first ApT, but the second ApT is in the correct position for strong cooperative interaction, rendering the third adenine hyperreactive to DEPC. This should then forbid binding to the third ApT but assist binding to the fourth, rendering the fifth adenine hyperreactive to DEPC, and so on. The same argument applies to the changes in DEPC modification seen with CGGC; binding to the CG step prevents binding to the first ApT on the 5'-side but assists interaction with the second ApT. Similarly binding to CpG will favour binding to the first ApT on the 3'-side of CGGC since this is separated from it by only two base pairs. The situation is less clear on the 5'-side of CCCG since all the adenines are equally reactive to DEPC, so that binding to each ApT step is equally likely. This suggests that the intervening CCC sequence does not participate in the cooperative process, possibly related to its inability to propagate structural changes. For the sequence CCGC cooperative effects will be less likely since the nearest ApT steps are separated from it by either one or three Nucleic Acids Research, Vol. 19, No. 24 6729 base pairs which are too close and too far respectively to allow efficient drug binding. With this fragment the alternations in DEPC modification are much less impressive, suggesting that the central drug molecule has less influence on the positioning of the remaining molecules. It is worth emphasising that in this case, and for the 5'-side of CCCG, the drug is interacting with the (AT). This is evidenced by the increased cleavage of the TpA bonds, seen even in the sequences lacking the central CpG step (see below). However, this interaction is random so that occupancy of each ApT step is equally probable. A precise explanation of the DNase I cleavage patterns is less easy, except to note that the most reactive ApT steps correspond to adenines which are less sensitive to modification by DEPC. According to the above model these bases are sandwiched by the drug molecule, suggesting that they remain in a more normal B-DNA configuration (they are not reactive to DEPC) and so are readily cut by DNase I. In contrast the structure of the intervening ApT steps must be altered by the unwinding caused by the drug molecule. The effects of echinomycin on (AT)15GG(AT)6 can only be explained by suggesting that the drug interacts at the central GG site. This can not be as strong as that at CpG since no clear DNAse I footprint is produced. However, it must be stronger than that at ApT since the central GG step is able to orient the drug molecules on the surrounding (AT)n. In contrast the fourfold repeat pattern is not produced with blocks of (AT), remote from canonical echinomycin binding sites, confirming that the pattern is a direct result of the juxtaposition of two sequences. Another possible echinomycin binding site at the centre of (AT)15GG(AT)6 might also be the TpG step. Interaction at TpG would leave three bases before the first available ApT on both sides and is therefore less likely to be cooperative, but would result in strong DEPC modification of the odd numbered adenines; this is indeed what we observe. Interaction at the symmetrically disposed GpA step would have the opposite effect, i.e. DEPC modification of the even adenines. We therefore suggest that the most likely secondary site for echinomycin on this fragment is at GG. If echinomycin can bind reasonably well to GG then why is no phasing observed for the fragments containing (AT)n blocks within the SmaI site (CCC/GGG), since these too are flanked by GG steps? In this case several overlapping GG steps are present; binding to each one will have opposite effects on the phasing pattern which should cancel each other out. The DNase I patterns observed for these fragments do indicate some interaction with echinomycin since cleavage of all the TpA steps is greatly enhanced. This could be due to either the drug binding randomly within the (AT)n blocks, or structural effects propagated by ligand interaction with the GG sites. It seems likely that both effects are occurring. Interaction at (ATT)n.(AAT). Both (ATT)4CG(AAT)4 and (TAA)4CG(TTA)4 showed only the three base pair cleavage pattern for DEPC or DNase I expected from the triplet nature of the sequences. No additional phasing was evident. Looking first at (ATT)4CG(AAT)4 binding to the central CpG must be weak (witness the poor DNase I footprint) yet is sufficient to produced strong DEPC modification at the first adenine (CGA). Four base pairs must separate this central site from the first available ApT step on both sides with an additional four base pairs before the next available ApT. It is therefore not surprising that no cooperative phasing effects are observed. No enhancements to DEPC modification are found in the ATT portion since every adenine is either part of the drug binding site (ApT) or separated from it by two base pairs. In the AAT portion the lower adenine (AAT) can either be sandwiched between the quinoxaline chromophores or located two bases to either side of a drug molecule bound to ApT. The upper adenine (AAT) is on the 5'-side of each ApT step (i.e.ATA). For the sequence (TAA)4CG(TTA)4 the first ApT steps are found two bases on either side of the central CpG. Although these secondary sites are as close as possible, avoiding neighbour exclusion, there is no stronger DEPC modification at these bases, suggesting that cooperative effects are not transmitted through the intervening AA and TT steps. Each of the available ApT sites is equally occupied. Model for binding at ApT These suggest that echinomycin can bind selectively to the dinucleotide ApT. The kinetics of this interaction are very different from that at CpG since no footprints are produced. What then is the basis of this sequence recognition? A clue to this may come from studies with the synthetic derivative TANDEM lacking the four N-methyl groups [16-19]. The crystal structure of this compound revealed that the peptide backbone adopted a different conformation due to the presence of two internal hydrogen bonds between the carbonyls of the alanines and the NHs of the valines [19]. As a result it was suggested that it could recognise the dinucleotide ApT by forming hydrogen bonds between the NHs of the alanines and the 2-keto groups of the thymines (instead of between the carbonyl group of alanine and the 2-amino group of guanine responsible for binding to CpG). Each of these potential groups is also present in echinomycin, though the crystal structure of the ligand suggests that they are in the wrong orientation. Binding to ApT would therefore require a change in the conformation of echinomycin. The energetics of such a conformational change are likely to depend on the nature of the cross-bridge and may explain why triostin A and echinomycin have different relative affinities for poly(dA-dT) and poly(dG-dC). This model predicts that a derivative lacking the NHs of alanine (i.e. [N-MeAla2, N-MeAla61 triostin A) should be unable to bind to ApT. Sequence selectivity As well as confirming the ability of echinomycin to bind selectively at CpG and demonstrating an additional interaction at ApT these results reveal the presence of other secondary sites at GG (CC) and weaker binding to the fragment (ATT)4CG (AAT)4 containing the central hexanucleotide TTCGAA. The latter is consistent with the results obtained with T15CGA15 [10] and suggests that the structure adopted by flanking regions of oligopyrimidines prevents efficient drug binding. Interaction at GG (CC) may arise from the formation of one specific hydrogen bond (to the second guanine) with the second symmetrically disposed potential bonding site on the molecule left unoccupied. This is similar to the ability of actinomycin (selective for GpC) to interact with other sequences of the type GpX (XpC) [20]. ACKNOWLEDGEMENTS This work was supported by grants from the Cancer Research Campaign and the Medical Research Council. 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