Interaction of echinomycin with An.Tn. and (AT)n regions flanking its CG binding site.

We have prepared DNA fragments containing the sequences A15CGT15, T15CGA15 and T(AT)8CG(AT)15 cloned within the SmaI site of the pUC19 polylinker. These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences. No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site. All the other fragments show the expected footprints around the CG site. Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3'-side of the CG become hyperreactive to diethylpyrocarbonate. Regions of alternating AT show unusual changes in the presence of the ligand. At low concentrations (5 microM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident. A similar pattern is seen with micrococcal nuclease. Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5'-direction across the two strands. We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT. DNase I footprinting experiments performed at 4 degrees C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are necessary consequence of drug binding.     

Echinomycin binding to alternating AT.

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.     

Structure of a pleiomeric form of poly d(AT):poly d(AT)

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.     

Homologous desensitization of ATP-mediated elevations in cytoplasmic calcium and prostacyclin release in human endothelial cells does not involve protein kinase C.

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.     

The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study.

The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.     

Proton NMR studies of [N-MeCys3,N-MeCys7]TANDEM binding to DNA oligonucleotides: sequence-specific binding at the TpA site.

[N-MeCys3,N-MeCys7]TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that [N-MeCys3,N-MeCys7]TANDEM binds strongly to DNA rich in A.T residues [Low, C. M. L., Fox, K. R., Olsen, R. K., & Waring, M. J. (1986) Nucleic Acids Res. 14, 2015-2033]. However, it was not known whether specific binding of [N-MeCys3,N-MeCys7]TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers [d(GGATATCC)]2 and [d(GGTTAACC)]2, [N-MeCys3,N-MeCys7]TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer [d(GGAATTCC)]2 as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that [N-MeCys3,N-MeCys7]TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of [N-MeCys3,N-MeCys7]TANDEM binding to DNA are discussed.     

Probing the conformations of eight cloned DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG.

The self complementary DNA dodecamers d(CGCGAATTCGCG), d(CGCGTTAACGCG), d(CGCGTATACGCG), d(CGCGATATCGCG), d(CGCAAATTTGCG), d(CGCTTTAAAGCG), d(CGCGGATCCGCG) and d(CGCGGTACCGCG) have been cloned into the Smal site of plasmid pUC19. Radiolabelled polylinker fragments containing these inserts have been digested with nucleases and chemical agents, probing the structure of the central AT base pairs. The sequences AATT and AAATTT are relatively resistant to digestion by DNase I, micrococcal nuclease and hydroxyl radicals, consistent with the suggestion that they possess a narrow minor groove. Nuclease digestion of TTAA is much more even, and comparable to that at mixed sequence DNA. TpA steps in ATAT, TATA and GTAC are cut less well by DNAse I than in TTAA. DNasel cleavage of surrounding bases, especially CpG is strongly influenced by the nature of the central sequence.     

DNA structure influences sequence specific cleavage by bleomycin.

We have examined the cleavage of several synthetic DNA sequences by iron(II)-bleomycin. We find that, although bleomycin cuts mixed sequence DNAs with a preference for GC = GT > GA >> GG, it efficiently cleaves regions of (AT)n cutting exclusively at ApT, not TpA. Isolated ApT steps show very little cleavage while blocks of three or more contiguous ATs are cut as efficiently as GpT. This cleavage is specific for (AT)n, since sequences of the type (TAA)n.(TTA)n and (ATT)n.(AAT)n are hardly cut at all. No cleavage is observed at ApC or CpA within sequences of the type (AC)n.(GT)n; regions of An.Tn are also not cut. Although the cobalt-bleomycin complex (which binds to but does not cleave DNA) yields good DNase I footprints at GT and GC sites, no footprints are observed within (AT)n, suggesting that although the cleavage reaction is efficient, the binding affinity is relatively weak. We propose a model in which bleomycin cleavage is determined by local DNA structure, while strong binding requires the presence of a guanine residue.     

Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities.     

Actinomycin D facilitates transition of AT domains in molecules of sequence (AT)nAGCT(AT)n to a DNAse I detectable alternating structure.

The interaction of actinomycin D with (AT)nAGCT(AT)n (where n = 2, 3, or 4) was investigated using a combination of imino proton NMR and DNAse I digestion. The stoichiometry of the interaction appears to be one:one with the actinomycin chromophore intercalated between the two GC base pairs. This binding event facilitates the conversion of the flanking repetitive AT regions to an alternating conformation characterized by induced sensitivity of the ApT sequences to attack by DNAse I. The neighboring TpA sequences do not exhibit rate changes as a function of binding of the drug. The potential relevance of such ligand induced DNA structural alterations is discussed.     

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.     

Effect of echinomycin on DNA methylation

Although echinomycin is reported to intercalate and to bind to DNA at CG dinucleotides, the effects of the drug on DNA methylation in vitro and in vivo are much less apparent than are the effects on DNA synthesis and cell growth.     

Towards echinomycin mimetics by grafting quinoxaline residues on glycophane scaffolds

Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferentially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of the compounds to calf-thymus DNA was studied using UV-vis and steady state fluorescence spectroscopy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomycin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found to be stable during 20ns simulations at 298K. However, the MD simulations of a glycophane complexed with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The distance between the quinoxaline residues in the glycophane at the end of the MD simulation was 7.3-7.5?, whereas in echinomycin, the distance between the residues was ～11?, suggesting that longer glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.     

Sequence-specific binding of luzopeptin to DNA.

We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.     

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.     

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.     

Footprinting of echinomycin and actinomycin D on DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine.

In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.     

DNA sequence recognition by under-methylated analogues of triostin A.

Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.     

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.     

DNA sequence recognition by the indolocarbazole antitumor antibiotic AT2433-B1 and its diastereoisomer

The antibiotic AT2433-B1 belongs to a therapeutically important class of antitumor agents. This natural product contains an indolocarbazole aglycone connected to a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-L-xylose. The configuration of the amino sugar distinguishes AT2433-B1 from its diastereoisomer iso-AT2433-B1. Here we have investigated the interaction of these two disaccharide indolocarbazole derivatives with different DNA sequences by means of DNase I footprinting and surface plasmon resonance (SPR). Accurate binding measurements performed at 4 and 25 degrees C using the BIAcore SPR method revealed that AT2433-B1 binds considerably more tightly to a hairpin oligomer containing a [CG](4) block than to an oligomer with a central [AT](4) tract. The kinetic analysis shows that the antibiotic dissociates much more slowly from the GC sequence compared to the AT one. Preferential binding of AT2433-B1 to GC-rich sequences in DNA was independently confirmed by DNase I footprinting experiments performed with a 117 bp DNA restriction fragment. The specific binding sequence 5'-AACGCCAG identified from the footprints was then converted into a biotin-labeled DNA hairpin duplex and compound interactions with this specific sequence were characterized by high resolution BIAcore SPR experiments. Such a combined approach provided a detailed understanding of the molecular basis of DNA recognition. The discovery that the glycosyl antibiotic AT2433-B1 preferentially recognizes defined sequences offers novel opportunities for the future design of sequence-specific DNA-reading small molecules.