Actinomycin D binds to metastable hairpins in single-stranded DNA.
We have examined the role of DNA composition in the binding of actinomycin D to single-stranded DNA. By using the fluorescent analogue 7-aminoactinomycin D, we were able to monitor binding of the drug to ssDNA with single base changes distant from the 5'-TAGT-3' site previously determined to be a high-affinity site for actinomycin D binding (Wadkins et al. (1996) J. Mol. Biol. 262, 53-68). Our binding studies indicated that secondary structures in the ssDNA were likely to be responsible for binding the drug. A series of six low-melting DNA hairpins containing all or part of the 5'-TAGT-3' binding site were synthesized. The highest Tm observed for the melting of these hairpins was 34.2 +/- 0.3 degrees C, and it depended on the length of the stem region. These metastable hairpins were stabilized by 7-aminoactinomycin D, with the drug shifting the Tm for the drug-hairpin complex to approximately 45 degrees C. The hairpins showed very high affinity (Kd approximately 0.1 microM) for 7-aminoactinomycin D, with some dependence on stem length. Digestion of the hairpins in the presence and absence of drug using mung bean nuclease, which specifically interacts with the loop region of hairpin DNA, revealed that the stable hairpins (i) contain a number of non-Watson-Crick base pairs, and (ii) undergo a conformational change in the loop region upon binding 7-aminoactinomycin D. Our results suggest that stabilization of unusual hairpins by actinomycin D may be an important aspect of the potent transcription inhibition activity of this drug.
Laboratory of Biophysical Chemistry, Cancer Therapy and Research Center, Institute for Drug Development, San Antonio, Texas 78245, USA. rwadkins@saci.org
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Wadkins
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Vladu
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Tung
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D015719:Single-Strand Specific DNA and RNA Endonucleases