Intra- and intermolecular tripler DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular tripler DNA assembly, We tested this hypothesis using the DNA tripler adopted by the murine c-myb protooncogene. The 5'-regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences, The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay, Gel mobility shift assays, nuclease and chemical hypersensitivity and SD-gel topological analyses as well as tripler-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular tripler structures in this sequence, Mithramycin binding within the tripler target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects, Molecular modeling studies further support these observations. Tripler-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal tripler structures compared to the non-treated and distamycin-treated cells, These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular tripler formation, as well as by physically preventing regulatory protein binding.