Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.
Wild-type p53 protein was shown to bind specifically to DNA sequences within SV40 (Bargonetti et al. 1991), the human ribosomal gene cluster (RGC) (Kern et al. 1991a), and the murine muscle creatine kinase gene (MCK) (Zambetti et al. 1992). However, a direct comparison of these three sites was not performed. Here we demonstrate, by filter binding and gel mobility-shift assays, that wild-type p53 binds with similar affinities to MCK and RGC sites but less tightly to the SV40 site. We examined the effects of two candidate regulators of p53 function, SV40 large T antigen and oncogenic mutant p53, on the binding of wild-type p53 to RGC DNA. We show that wild-type T antigen prevents p53 from binding to the RGC site under all conditions tested. Moreover, two temperature-sensitive mutant SV40 T antigens, which fail to transform cells at the nonpermissive temperature, prevent p53 from binding to the RGC site at the permissive, but not at the restrictive, temperature. The ability of complexes containing wild-type p53 and tumor-derived mutant p53 proteins to bind to RGC DNA varies according to the position of the mutation. Complexes containing wild-type and either his175 or his273 mutant p53 proteins are completely unable to bind to the RGC DNA sequence. Interestingly, a complex containing wild-type p53 and the trp248 mutant p53 characteristic of Li-Fraumeni syndrome patients displays nearly wild-type levels of binding. Perhaps this mutant allele can be tolerated in these individuals because the wild-type mutant p53 complex maintains the ability to bind to DNA. Our data indicate that the oncogenic potential of both T antigen and some mutant p53 proteins is the result of their ability to block binding of wild-type p53 to DNA.
Bargonetti
J
J
Department of Biological Sciences, Columbia University, New York, New York 10027.
Reynisdóttir
I
I
Friedman
P N
PN
Prives
C
C
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D016172:DNA Fingerprinting; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D012407:rRNA Operon / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D009841:Oligonucleotides; D016159:Tumor Suppressor Protein p53; D004247:DNA; D003402:Creatine Kinase