INTERACTION OF P53 WITH ITS CONSENSUS DNA-BINDING SITE

We have analyzed the specific interaction of murine p53 with the conse nsus DNA-binding sequence 5'-AGACATGCCT-AGACATGCCT-3'. We used segment s of p53 lacking the C-terminal, nonspecific DNA-binding domain becaus e the presence of an autonomous nonspecific DNA-binding domain in wild -type p53 would complicate analysis of site-specific DNA binding. p53 amino acids 1 to 360 bind the consensus sequence as tetramers, and DNA binding promotes tetramer-tetramer interactions. p53 amino acids 80 t o 290, lacking both the nonspecific DNA-binding and tetramerization do mains, consistently bind consensus DNA as four monomers and only as fo ur monomers. The virtual absence of stable binding by fewer than four monomers, even at low concentrations of p53, argues that binding by am ino acids 80 to 290 is strongly cooperative. Because p53 tetramers and monomers do not simultaneously bind a single DNA consensus sequence, we conclude that a single tetramer of wild-type p53 engages the recogn ition sequences of the entire DNA consensus site. We further show that consensus DNA consists of two functional half-sites. Insertions, dele tions, or rearrangements within the half-sites reduce DNA binding dram atically. In contrast, two half-sites separated by insertions bind p53 relatively efficiently. Insertions that place half-sites on opposite faces of the DNA helix reduce DNA binding more than insertions that pl ace half-sites on the same face of the helix. Transcription studies, i n vivo, strongly confirm the rotational specificity of the p53 interac tion with consensus DNA. The ability of single p53 tetramers to bind s eparated DNA half-sites argues that p53 has a flexible tetramerization region.