Regulation of mutant p53 temperature-sensitive DNA binding.
We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 --> Ala, Arg-175 --> His, Arg-248 --> Trp, Arg-249 --> Ser, and Arg-273 --> His). While all mutants were defective for binding to DNA at 37 ;C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33 ;C), and several mutants activated transcription from a p53-responsive promoter at 26 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37 degrees C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.
Friedlander
P
P
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Legros
Y
Y
Soussi
T
T
Prives
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D016158:Genes, p53; D007700:Kinetics; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D018411:Spodoptera; D013696:Temperature; D013816:Thermodynamics; D015533:Transcriptional Activation; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA