Dissociation Kinetics of Echinomycin from CpG Binding Sites in Different Sequence Environments

Abstract

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.

Antitumor Activity, X-ray Crystal Structure, and DNA Binding Properties of Thiocoraline A, a Natural Bisintercalating Thiodepsipeptide

“…Taken together, these results appear to indicate that flanking sequences also have some bearing on the sequence selectivity of thiocoraline, in good consonance with the more limited footprinting data, with sites possessing an A on the 5′ side being more stabilized, and this is also true for echinomycin. For this latter drug, the greater ∆T m for ACGT compared to TCGA is in nice agreement with earlier results showing faster dissociation kinetics from TCGA than from ACGT when the CpG site is found in an alternating AT environment 24 whereas the effect on ACGA is more concentration-dependent. The most striking difference between the profiles obtained for these two drugs, however, lies in the fact that a ∆T m plateau is reached for thiocoraline at the highest concentrations tested but not for echinomycin.…”

“…The Δ T m values reported in Table and Figures −8 correspond to T m2 − T m1 . Drug dissociation from DNA takes place in a single-step reaction, which implies that both chromophores are simultaneously withdrawn from the DNA lattice. …”

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

“…Novel analogues of the triostins were produced by supplementing S. triostinicus cultures with a variety of haloor amino-substituted quinoxaline-2-carboxylic acids, which were incorporated as the chromophores. 67, 68 Additionally, a quinoline and a 6-fluoro-substituted quinoxaline analogue were generated by supplementing with quinoline-2-carboxylic acid and DL-5-fluorotryptophan respectively (28,29). [68][69][70] This last discovery prompted the suggestion that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the triostin ring system.…”

The UvrA‐like protein Ecm16 requires ATPase activity to render resistance against echinomycin

“…One such antibiotic produced by Streptomyces is echinomycin, the first identified DNA bisintercalator (Waring, 1992). DNA intercalators bind reversibly to the DNA double helix in a sequence independent manner by inserting one or more planar, fused aromatic ring groups in between adjacent base pairs (Fletcher & Fox, 1996). Echinomycin, a member of the quinomycin family of antibiotics, contains two quinoxaline chromophores attached to a cyclic octadepsipeptide (Chen & Patel, 1995; Dawson et al, 2007; Dell et al, 1975) (Figure 1a).…”