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.
Papoulas
O
O
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Williams
N G
NG
Kingston
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells; D005814:Genes, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D004247:DNA