Unstable Hoogsteen base pairs adjacent to echinomycin binding sites within a DNA duplex.

Abstract

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...

Widespread transient Hoogsteen base pairs in canonical duplex DNA with variable energetics

“…To more broadly examine the occurrence and sequence-specificity of transient HG bps in canonical duplex DNA, we carried out 13 C and 15 N R 1ρ NMR RD measurements targeting sugar C1′ and base C6/8 or N1/3 resonances in 20 A•T and 13 G•C bps in eight DNA duplexes that encompass a variety of sequence motifs, including (A•T) n repeats of varying length (n=2, 4, 5 and 6), a (CA) 3 repeat, a duplex sequence that forms HG bps upon binding to the antibiotic echinomycin, 1,26,27 a (CG) 3 repeat capable of forming Z-DNA, 28 and a B/Z junction forming sequence 29,30 (Fig. 1b).…”

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

“…The present study of a dumbbell shows similar buckling of the closing base pair, caused by translating timeaveraged NOE data directly into distances with neglect of equilibria between anti and syn forms. These observations raise doubts about the presence of pure syn rotamers instead of syn/high-anti/anti equilibria (at low tempera- (Kallick and Wemmer, 1991) and in echinomycin-DNA complexes (Gilbert et al, 1989;Gilbert and Feigon, 1992). Moreover, temperature-dependent line broadening was found specifically for the HI' signal of dG(10) in an RNA hairpin 5'-r(GGGCGC-UGGG-ACGCCCGUC)-3', containing the CUGGGA loop of the HIV-1 TAR element (Colvin et al, 1993).…”

Bisintercalator natural products with potential therapeutic applications: isolation, structure determination, synthetic and biological studies

“…32 In several NMR-based studies, Feigon and co-workers showed that while Hoogsteen base-pairing may occur, it tended to be at the ends of the oligonucleotides studied and internal base-pairs were dynamic, alternating between open, WatsonCrick or Hoogsteen forms. 33 More recent crystal structures have again demonstrated the formation of Hoogsteen base-pairs. 34 Feigon's NMR studies also demonstrated that differences in sequence can lead to variations in cooperativity of binding.…”