CHARACTERIZATION OF HIGH AFFINITY ACTINOMYCIN D BINDING TO EUKARYOTIC DNA

Actinomycin D in low concentrations was suggested to inhibit ribosomal RNA (rRNA) transcription via an extranucleolar mechanism. Actinomycin D was proposed to inhibit unique messenger RNAs (mRNAs) coding for proteins needed for the maintenance of rRNA transcription. According to this hypothesis actinomycin D would bind to specific nonribosomal DNA with high affinity. This hypothesis was investigated by isolating high molecular weight rat liver DNA, digesting it with restriction endonuclease EcoRI, adding [³H] actinomycin D in low concentration, performing RPC-5 chromatography to separate the restriction fragments and subsequent hybridization to rRNA. It was observed that actinomycin D bound to nonribosomal DNA with high affinity. The same experiment was performed with nucleolar DNA. High affinity actinomycin D binding was not observed in nucleolar DNA. Discrete high affinity binding DNA for actinomycin in rat liver DNA was also observed when another restriction endonuclease BamHI was used to cleave rat liver DNA. However, with rat liver DNA digested with restriction endonuclease HindIII, such a high affinity actinomycin D binding DNA was not observed. Actinomycin D was also demonstrated to bind to discrete site(s) in at least four more eukaryotic species (salmon, calf, herring and human) after DNA from these species were digested by EcoRI, labeled actinomycin D added, and RPC-5 chromatography performed. Labeled actinomycin D bound to its high affinity binding DNA was displaced by unlabeled actinomycin D in a concentration range of biological significance. However, six other antitumor agents, (doxorubicin, aclacinomycin, carminomycin, marcellomycin, musettamycin and pyrromycin) which also intercalate into DNA, did not significantly displace labeled actinomycin D from its high affinity binding DNA. Since this high affinity actinomycin D binding DNA is hypothesized to be involved in the inhibition of rRNA transcription, the actinomycin D binding DNA could have a role in the regulation of rRNA transcription. To date this is the first time that a probable regulatory DNA has been characterized by selective drug binding