Interaction of p53 with Its Consensus DNA-Binding Site

Abstract

We have analyzed the specific interaction of murine p53 with the consensus DNA-binding sequence 5-AGACATGCCT-AGACATGCCT-3. We used segments of p53 lacking the C-terminal, nonspecific DNA-binding domain because 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 to 290, lacking both the nonspecific DNA-binding and tetramerization domains, consistently bind consensus DNA as four monomers and only as four monomers. The virtual absence of stable binding by fewer than four monomers, even at low concentrations of p53, argues that binding by amino 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 recognition sequences of the entire DNA consensus site. We further show that consensus DNA consists of two functional half-sites. Insertions, deletions, or rearrangements within the half-sites reduce DNA binding dramatically. 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 place half-sites on the same face of the helix. Transcription studies, in vivo, strongly confirm the rotational specificity of the p53 interaction with consensus DNA. The ability of single p53 tetramers to bind separated DNA half-sites argues that p53 has a flexible tetramerization region.Specific DNA binding and transcriptional activation play central roles in the suppression of cellular proliferation by p53 (6,29,32). Most p53 mutations that are associated with human cancer affect the central conserved region of p53 (13) that is necessary and sufficient for specific DNA binding (2, 17, 31). Indeed, many mutations severely reduce site-specific DNA binding (1, 15) and transactivation in vivo (9,16,20). In model systems, there is also an excellent correlation between the transactivation and suppression functions of p53 (19,21). Removal of the natural transactivation domain of p53 blocks suppression of transformation by other oncogenes. Replacement of the transactivation domain with the heterologous VP16 transactivation domain restores transactivation and suppression.Studies of human and murine p53 have identified a number of autonomous functional domains. The N-terminal, acidic region strongly activates transcription when positioned at a promoter either as part of p53 or as a chimera with the GAL4 DNA-binding domain (10,20). The large central conserved region of p53 binds specific DNA sequences (2, 17, 31) and forms unstable oligomers without a preference for a particular oligomeric form (30). The C-terminal region assembles stable tetramers (30) and binds to DNA without apparent specificity (31). Surprisingly, C-terminal-truncation mutants lac...

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

“…This model can explain, and bring together, several seemingly contradictory observations, e.g. that in some cases at least two half-sites were shown to be required for binding (ElDeiry et al, 1992;, while in others the presence of a single half-site was su cient for binding Wang et al, 1995b), or the requirement for carboxy-terminal activation of the p53 protein for binding to some, but not to all target sites.…”

p53-Induced DNA Bending: The Interplay between p53−DNA and p53−p53 Interactions

“…Biophysical and biochemical data 11,[21][22][23][24] were obtained and a similar p53 core domain tetramer-DNA complex model was proposed 11,[21][22][23][24] . The specific ways of association between p53 and DNA and between p53 monomers are supported by experiments 18,[25][26][27] .…”

Transcriptional regulation and induction of apoptosis: implications for the use of monomeric p53 variants in gene therapy

“…In vitro DNA binding of monomeric p53 can also be achieved by increasing the concentration of the protein in the binding reaction, yielding complexes migrating similarly to wild-type p53-DNA complexes (data not shown). 54 Monomeric p53 variants with a functional DNA binding domain probably aggregate on p53 response elements to form labile DNAprotein complexes. Such complexes are still able to initiate the transcription of genes in vivo.…”

“…We suggest that the efficiency of DNA binding of p53 is limiting the transcriptional induction and consider this to be the reason for the more effective transactivation by wild-type p53. 19,[54][55][56][57] At high concentrations of wild-type p53 transactivation activity decreased again. p53 functions as a transcription factor itself and contacts members of the basal transcription machinery, such as TBP, the TATA binding protein 58 and the co-activator p300.…”