Mithramycin inhibits transcription by binding to G/Crich
sequences, thereby preventing regulatory protein
binding. However, it is also possible that mithramycin
inhibits gene expression by preventing intramolecular
triplex DNA assembly. We tested this hypothesis using
the DNA triplex adopted by the murine cmyb protooncogene.
The 5regulatory region of cmyb contains
two polypurine:polypyrimidine tracts with imperfect
mirror symmetry, which are highly conserved in the
murine and human cmyb 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 2Dgel topological analyses
as well as triplexspecific antibody binding studies
confirmed the formation of purine*purine:pyrimidine
inter and pyrimidine*purine:pyrimidine intramolecular
triplex structures in this sequence. Mithramycin
binding within the triplex target site displaces the major
groovebound oligonucleotide, and also abrogates
the supercoildependent HDNA formation, whereas
distamycin binding had no such effects. Molecular
modeling studies further support these observations.
Triplexspecific antibody staining of cells pretreated
with mithramycin demonstrates a reversal of chromosomal
triplex structures compared to the nontreated
and distamycintreated cells. These observations
suggest that DNA minor groovebinding drugs interfere
with gene expression by precluding intramolecular
triplex formation, as well as by physically preventing
regulatory protein binding.
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