DNA binding activities of c-Myc purified from eukaryotic cells.

c-Myc is a nuclear phosphoprotein which contains both a leucine zipper and a helix-loop-helix dimerization motif. These are adjacent to a basic region believed to make specific contacts with DNA upon dimerization. We report the purification of full-length c-Myc to near homogeneity from two independent eukaryotic systems: the baculovirus overexpression system using an insect cell host, and Chinese hamster ovary cells containing heat-inducible c-myc genes. The DNA binding capabilities of these preparations were characterized. Both preparations contain two distinct activities that bind specifically to sequences with a core of CACGTG. The Myc protein is solely responsible for one of these binding activities. Specific sequences that bound to c-Myc were selected from a large pool of random DNA sequence. Sequencing of individual binding sites selected by this procedure yielded a 12-base consensus, PuACCACGTGCTC, for c-Myc binding. Both protein preparations additionally demonstrated a distinct complex, containing both c-Myc and a copurifying 26-29-kDa protein, that bound to DNA with higher affinity than Myc alone. Selection of specific DNA sequences by this complex revealed a consensus binding site similar to the 12-base consensus described above. These data demonstrate that c-Myc isolated from eukaryotic cells is capable of sequence-specific DNA binding and further refine the optimal sequence for c-Myc binding. These protein preparations should prove useful in further characterizing the biochemical properties of c-Myc.