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.
Shen
J
J
Department of Molecular and Cellular Oncology, Box 79, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA.
Wang
J C
JC
Van Dyke
M W
MW
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003609:Dactinomycin / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism
D004268:DNA-Binding Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D009841:Oligonucleotides; D003609:Dactinomycin; D004247:DNA; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I