DNA-binding domain of human c-Myc produced in Escherichia coli.
We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.
Dang
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CV
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
van Dam
H
H
Buckmire
M
M
Lee
W M
WM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D013205:Staphylococcal Protein A / Q000235:genetics
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004247:DNA; D003488:Cyanogen Bromide