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

Abstract

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 μM actinomycin. A χ 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 258 nM (average 98 nM). 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.