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
J. Bargonetti et al., 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, Genes & development, 7(12B), 1993, pp. 2565-2574
p53 is a sequence-specific DNA-binding oligomeric protein that can act
ivate transcription from promoters bearing p53-binding sites. Whereas
the activation region of p53 has been identified within the amino term
inus, the location of the specific DNA-binding domain has not been rep
orted. Thermolysin treatment of p53 protein generates a stable proteas
e-resistant fragment that binds with marked specificity to p53 DNA-bin
ding sites. Amino-terminal sequencing of the fragment located the ther
molysin cleavage site to residue 91. Because the fragment does not con
tain the cdc2 phosphorylation site at Ser-315, we conclude that the th
e site-specific DNA-binding domain of p53 spans the central region of
the protein. The vast majority of the mutations in oncogenically deriv
ed p53 proteins are located within this central portion of the molecul
e. Such mutant p53 proteins exhibit defective sequence-specific DNA-bi
nding. Although thermolysin digestion of mutant p53 proteins generates
proteolytic patterns that differ from wild-type protein, one mutant t
ested, His-273, generates a resistant' fragment that migrates with a s
imilar electrophoretic mobility to the wild-type protease-resistant fr
agment. Interestingly, although intact mutant His-273 protein binds to
DNA at 20-degrees-C, the thermolysin-resistant mutant fragment does n
ot. 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 contrib
ute to both nonspecific DNA-binding and oligomerization, they are not
required for sequence-specific DNA-binding.