Actinomycin D is an intercalating antibiotic/antitumor agent which binds in an equilibrium fashion to DNA. The drug exhibits a pronounced specificity for 5'-GC-3' sequencies. The in vivo efficacy of the drug has been reputed to be a function of DNA binding. Intercalating agents such as actinomycin D have also been shown to impose well defined DNA secondary structure changes at their intercalation site. In the research outlined in this proposal the DNA secondary structure altering properties of actinomycin, viewed as a model intercalater, will be examined on a series of synthetic DNA duplexes enzymatically. Preliminary experiments have shown that the endonuclease DNAse I is ideally suited for this task having the ability to both locate actinomycin on DNA molecules and to respond to alteration in DNA secondary structure induced by ligand binding. By taking into account known structure: sequence motifs it will be possible to assess at single base-pair resolution how structural information is propagated through a DNA double helix. Specifically, DNAse I cleavage rates will be examined on four model B DNA duplexes to generate information on the ability of the enzyme to accurately report local helical twist values. This information will be utilized in experiments employing 'altered' DNA sequences in the absence of actinomycin D to evaluate the basal structural determinates of the sequences employed. The same sequences will then be analyzed in the presence of an actinomycin D bound in juxtaposition to the altered DNA structures. In this way it is possible to assess the secondary structure effects of intercalative DNA binding. The fact that topoisomerases which are sensitive to alterations in DNA secondary structure respond to intercalating molecules in vivo points to this type of structural response as important in the future design of new, clinically important, anti- cancer agents.
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