Actinomycin D binds with highest affinity to nonribosomal DNA

Based on previous evidence (T.J. Lindell (1976) Nature 263, 347-350 and T.J. Lindell, A.F. O'Malley, and B. Puglisi (1978) Biochemistry 17, 1154-1160) it was anticipated that actinomycin D in a low concentration would bind to DNA which is involved in the regulation of rRNA transcription in eukaryotes. This was examined by digestion of rat (liver) DNA with EcoRI, adding [3H]actinomycin D (0.004 microgram/ml) and resolution of the restriction fragments by RPC-5 chromatography. Labeled actinomycin D was found to bind with highest affinity to a single fraction consistently eluting at 0.63 M KCl. A similar digestion and fractionation of nucleolar DNA, which is enriched in rDNA sequences, did not reveal a similar high affinity binding of actinomycin D. This fraction of DNA which binds actinomycin D with highest affinity did not contain any rDNA as evidenced by hybridization with [32P]rRNA. Therefore, at the concentration of 0.004 microgram/ml, actinomycin D does not selectively bind to rDNA as evidenced by two independent criteria. Whether this high affinity actinomycin D-binding DNA has any function in the regulation of rRNA transcription remains to be determined.