Determination of the DNA sequence recognized by the bHLH-zip domain of the N-Myc protein

The DNA-binding domain of the murine N-Myc protein, comprising the basic helix-loop-helix-zipper (bHLH-zip) region was expressed as a fusion protein in E. coli. The affinity purified glutathione-S-transferase-N-Myc fusion protein (GST-N-MYC) was used to select the N-Myc specific DNA-recognition motif from a pool of random-sequence oligonucleotides. After seven rounds of binding-site selection, specifically enriched oligonucleotides were cloned and sequenced. Of 31 individual oligonucleotides whose sequences were determined, 30 contained a common DNA-motif, defining the hexameric consensus sequence CACGTG. We confirm by mutational analysis that binding of the N-Myc derived bHLH-zip domain to this motif is sequence-specific.