BINDING OF ACTINOMYCIN-D TO THE T(G)(N)T MOTIF OF DOUBLE-STRANDED DNA - DETERMINATION OF THE GUANINE REQUIREMENT IN NONCLASSICAL, NON-GPC BINDING-SITES

Strong binding of the antitumor antibiotic actinomycin D to the sequence 5'-TGGGT-3' in double-stranded DNA was recently established by equilibrium binding studies (Bailey et al., 1993). Actinomycin D binding to this -TGGGT- containing sequence was shown to be comparable to that of an -XGCY- containing oligonucleotide (K-a similar to 10(6) M(-1)). Investigation of -TGGGT- as a high-affinity binding site for actinomycin D follows from our 1989 sequencing study (Rill et al., 1989) in which the photoaffinity analog of actinomycin D (7-azidoactinomycin D) was used to determine DNA base sequence specificities and neighboring base effects. The studies presented here examine the guanine requirements for actinomycin D binding to such nonclassical (non-dGpC) sites by varying the number of central guanine residues in a series of selected duplex oligonucleotides. The central -T(G),T- motif varies from n equals 1 to 4. Actinomycin D binding to each of these undecamers is characterized and correlated with binding to oligonucleotides of identical length and similar sequences that contain classical dGpC binding sites. Binding affinities of actinomycin D to this series of oligonucleotide duplexes (10 degrees C) can be summarized as -TGGGT- > -TGGT- > -TGGGGT- > -TGT. The kinetics of SDS-induced dissociation of actinomycin D from these oligonucleotides reveal single-exponential decays with duration dependent on the sequence at the binding site. With the exception of the -TGGGT-containing oligomer, dissociation times for the T(G)(n)T duplexes were drastically different and much shorter than times obtained for the dissociation of actinomycin D from oligonucleotides having classical dGpC sites. Dissociation constants ranged from 1463 s for -TGGGT- to 67 a for -TGGT-. The binding energetics for these drug-DNA interactions are quantitated and are presented as a function of the number of central guanines. We observe an unusual (positive) binding enthalpy for the interaction of actinomycin D with the -TGGT- sequence, whereas binding to all of the other sequences exhibits negative enthalpies. Thus, addition or deletion of one G to or from the original -TGGGT- site causes significant changes in both binding affinities and energetics, even though the number of binding sites per duplex remains constant. These changes and how they correlate with changes in the base sequence are presented as a full thermodynamic profile.