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The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.

The bisintercalation complex present between the DNA octamer [d(ACGTACGT)12 and the cyclic octadepsipeptide antibiotic echinomycin has been studied by one-and two-dimensional proton NMR, and the results obtained have been compared with the crystal structures of related DNA-echinomycin complexes. Two echinomycins are found to bind cooperatively to each DNA duplex at the CpG steps, with the two quinoxaline rings of each echinomycin bisintercalating between the C-G and A-T base pairs. At low temperatures, the ANT base pairs on either side of the intercalation site adopt the Hoogsteen conformation, as observed in the crystal structures. However, as the temperature is raised, the Hoogsteen base pairs in the interior of the duplex are destabilized and are observed to be exchanging between the Hoogsteen base pair and either an open or a Watson-Crick base-paired state. The terminal AT base pairs, which are not as constrained by the helix as the internal base pairs, remain stably Hoogsteen base-paired up to at least 450C. The implications of these results for the biological role of Hoogsteen base pairs in echinomycin-DNA complexes in vivo are discussed.Echinomycin is a cyclic octadepsipeptide antibiotic with two quinoxaline rings (Structure 1) that bisintercalate into DNA (1, 2). The drug is a potent antitumor and antimicrobial agent (1, 2). Cleavage inhibition patterns ("footprinting") with DNases I and 11 (3) and methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] (4) showed that the preferred binding sites are centered around a CpG step. The MPE-Fe(II) studies showed that the binding site is 4 base pairs and that the strongest binding sites are TCGT and ACGT. The DNase and MPEFe(II) studies also revealed that A-T base pairs adjacent to the binding site (and in some cases, runs of A-T base pairs distal to the binding sites) are more sensitive to cleavage by these reagents than is uncomplexed DNA, suggesting that these regions might have an altered conformation from B-DNA. Crystal structures of echinomycin and the related triostin A with the DNA hexamer [d(CGTACG)]2 have been solved (5, 6). Two echinomycins are found to bind to each DNA duplex, with the echinomycin rings bracketing the CpG steps and the polypeptide arranged in the minor groove. A remarkable feature of these structures is that the A-T base pairs are Hoogsteen base-paired (7) with the adenines in the syn conformation.More recently, Mendel and Dervan (8) demonstrated that echinomycin binding results in enhanced sensitivity to diethyl pyrocarbonate (EtOOC)20 of purines adjacent and distal to echinomycin-binding sites. These results were presented as being consistent with Hoogsteen base-pair formation as a result of drug binding in these longer DNA fragments. Waring and co-workers (9) have also investigated this enhanced sensitivity of (EtOOC)20 to purines in echi- nomycin-bound DNA, but they conclude that the evidence does not support the formation ofHoogsteen base pairs as the sole cause of enhanced sensitivity to (EtOOC)20 in the drug-DNA complexes.We have i...

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Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.