Footprinting of echinomycin and actinomycin D on DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine.
In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.
Jennewein
S
S
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK.
Waring
M J
MJ
D015075:2-Aminopurine / Q000031:analogs & derivatives*; D001482:Base Composition; D001665:Binding Sites; D003596:Cytosine; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D017931:DNA Primers; D003609:Dactinomycin / Q000737:chemistry*; D004448:Echinomycin / Q000737:chemistry*; D006860:Hydrogen Bonding; D019271:Hypoxanthine; D007288:Inosine; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013941:Thymine
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D003609:Dactinomycin; D019271:Hypoxanthine; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D004448:Echinomycin; D007288:Inosine; D003596:Cytosine; D004247:DNA; D013941:Thymine