Intra- and Intermolecular Triplex DNA Formation in the Murine c-myb Proto-Oncogene Promoter Are Inhibited by Mithramycin

Nadarajah Vigneswaran, Jeyasakthy Thayaparan, Judy Knops, John Trent, Vladimir Potaman, Donald M. Miller and Wolfgang Zacharias

Abstract

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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Biological Chemistry keeps you up-to-date with the latest advances in the molecular life sciences. The journal publishes Research Articles, Short Communications, Reviews and Minireviews. Areas include: general biochemistry/pathobiochemistry, structural biology, molecular and cellular biology, genetics and epigenetics, virology, molecular medicine, plant molecular biology/biochemistry and novel experimental methodologies.

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