A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein.
p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20 degrees C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.
Bargonetti
J
J
Department of Biological Sciences, Columbia University, New York, New York 10027.
Manfredi
J J
JJ
Chen
X
X
Marshak
D R
DR
Prives
C
C
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D009841:Oligonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013820:Thermolysin; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D009841:Oligonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D013820:Thermolysin