echinomycin and 2--deoxytubercidin

In order to investigate the possible importance of Hoogsteen base pairing to the DNA-binding ability of echinomycin, quantitative DNase I footprinting has been performed. The substrate was the tyrT DNA restriction fragment, either "native" or substituted with one of the purine analogs 2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine in both strands. The modified DNA species were prepared by PCR and selectively labeled at the 5' terminus of one strand (usually the upper "Watson" strand) with [32P]ATP and polynucleotide kinase. Proper incorporation of the analog nucleotides was verified by Maxam-Gilbert G- and C-sequencing reactions as well as exposure to osmium tetroxide and diethyl pyrocarbonate. OsO4 was found to react strongly with the 7-deaza nucleotides, providing a good check of faithful incorporation. The previously observed echinomycin-induced hyperreactivity of purines toward diethyl pyrocarbonate was eliminated by incorporating the appropriate 7-deazapurine. The DNase I footprinting titration studies greatly refined the existing knowledge of the DNA-binding characteristics of echinomycin, as they revealed five general types of concentration-dependent behavior at single-bond resolution. Estimates of microscopic binding constants at individual DNA binding sites were obtained by measuring the antibiotic concentration which produced a half-maximal effect on the concentration of a given DNase I cleavage product. All binding sites contained one or more CpG steps, and all CpG steps analyzed formed part of a binding site for echinomycin. No consistent differences in the estimated binding constants for these sites were observed by comparing normal and modified DNAs, indicating that the abolition of formal Hoogsteen pairs did not significantly alter the thermodynamics of echinomycin-DNA interaction. The lack of any detectable decrease in binding constants for critical sites in the 7-deazapurine-substituted DNAs argues against any anti-syn conformational transition of purine nucleosides occurring in association with the bis-intercalative complex formation.

Topics: Amino Acid Sequence; Analysis of Variance; Base Composition; Base Sequence; Deoxyguanosine; Deoxyribonuclease I; DNA, Bacterial; Echinomycin; Genes, Bacterial; Molecular Sequence Data; RNA, Transfer, Tyr; Structure-Activity Relationship; Tubercidin

To show conclusively that the critical structural deformation of double-helical DNA that is induced by the binding of quinoxaline antibiotics does not involve the formation of Hoogsteen base pairs, we have prepared a DNA fragment containing the nucleoside analog 7-deaza-2'-deoxyadenosine in one of the two strands. This DNA fragment was subjected to treatment with the thymidine-specific reagent osmium tetroxide and to DNase I "footprinting" in the presence or absence of micromolar concentrations of echinomycin. We report that this anti-tumor antibiotic binds to DNA containing the nucleoside analog as well as to natural DNA and that the previously reported hypersensitivity to osmium tetroxide of certain thymidine residues adjacent to echinomycin binding sites is maintained in analog-containing DNA. Since these thymidines are rendered incapable of participating in Hoogsteen base pairs by the incorporation of 7-deaza-2'-deoxyadenosine, we conclude that this unusual base-pairing scheme is not the cause of the observed hypersensitivity to osmium tetroxide and that it therefore results from a large local unwinding of the DNA in the presence of the antibiotic. Moreover, preventing the possibility of Hoogsteen base pairing does not preclude echinomycin binding.

Topics: Aminoglycosides; Anti-Bacterial Agents; Base Composition; Base Sequence; Deoxyribonuclease I; DNA; DNA, Single-Stranded; Echinomycin; Molecular Sequence Data; Molecular Structure; Nucleotide Mapping; Oligodeoxyribonucleotides; Osmium; Osmium Tetroxide; Plasmids; Promoter Regions, Genetic; Quinoxalines; Terminator Regions, Genetic; Tubercidin