Examining Flanking Sequence Specificity and Topological Specificity in the Binding of Various Molecular Types to DNAs Using Restriction Endonuclease Activity Assays

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Observing alteration of restriction enzyme activity has been employed frequently to determine the sequence specificity of the binding of many types of molecules to DNAs. Generally, these studies employed restriction enzymes which cut the target DNA several times. The effects of binding to sequences flanking the restriction enzyme cleavage sites may have been obscured. In this study, we report on restriction enzyme activity assays of the binding of the intercalators actinomycin D, ametantrone and ethidium, the groove binder netropsin, and the covalent binding cisplatin to a mixture of supercoiled and relaxed phiX 174 RF DNA using restriction enzymes which cleave this DNA once or twice. Sequence selectivities and topological selectivities were observed for these ligands. In some cases restriction enzymes not containing the reported preferred binding sites had altered activities, suggesting binding to flanking sequences affects activity in neighboring DNA sequences.