Chemical probes reveal no evidence of Hoogsteen base pairing in complexes formed between echinomycin and DNA in solution

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Abstract
Five different DNA fragments have been treated with a range of conformationally sensitive reagents in an effort to probe structural chages in DNA associated with binding of the bis-intercalating antibiotic echinomycin. For each probe, the intensity and pattern of its reactivity with DNA have been analyzed in order to elucidate the effect of antibiotic binding on the accessibility of a specific site of sites to chemical attack. It was found that in one of the DNA fragments, pTyr2DNA, several purine residues exhibit enhanced reactivity to diethyl pyrocarbonate (DEPC) in the absence of bound antibiotic, and that this strongly sequence specific reaction is enhanced in the presence of quite low echinomycin concentrations. The echinomycin-dependent reactivities towards DEPC of three homologous DNA fragments, chosen for their subtly different antibiotic binding characteristics, were also investigated. It was found that small changes in base sequence generate striking changes in susceptibility to modification by DEPC. The abolition of one antibiotic binding site leads to the creation of an new, intense DEPC-reactive site. In the presence of moderate concentrations of echinomycin, specific thymidine residues exhibit enhanced reactivity towards osmium tetroxide. No differences in the reactivities of the DNA fragments towards bromoacetaldehyde, S1 nuclease, dimethyl sulphate of potassium tetrachloropalladinate were observed in the presence of the antibiotic. DEPC reactions were performed on tubercidin (7-deaza-adenosine) to determine the DEPC reactive positions in situations where N-7 is inaccessible. Tubercidin was found to be generally resistant to attack by DEPC followed by treatment with base. We conclude that the bulk of structural changes induced by the binding of echinomycin to DNA do not involve Hoogsteen base pairing, but rather are due to sequence-specific unwinding of the helix in a manner which is strongly dependent on the nature of surrounding nucleotide sequences.