Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA.

An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 microM actinomycin. A chi(2) statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5'-(T/A)GC(A/T)-3', giving a p<0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258nM (average 98nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5'-TGCTGCT-3'), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiological conditions.