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Loss of transactivation and transrepression function, and not RPA binding, alters growth suppression by p53
L M Leiter1, J Chen, T Marathe
1Division of Molecular Oncology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
The tumor suppressor protein p53 activates transcription from promoters with specific p53 binding elements, represses transcription from promoters without such elements and interacts with and inhibits the single-stranded DNA binding activity of the human DNA replication factor RPA. All these activities involve the N terminal 70 amino acids of p53. Dissection of the domains of p53 which bind RPA suggest that multiple sub-domains of the protein synergize to give strong RPA binding. Point-mutations in one of these sub-domains of p53 significantly diminish its ability to interact with RPA. A multimer of a peptide from p53 which includes these residues, or of a peptide from the acidic activation domain of the prototypic trans-activator protein VP16, can itself bind to RPA. Comparison of sequences of these multimeric peptides suggests that aromatic amino acids flanked by negatively charged residues are important for binding RPA. Several alleles of p53 with point mutations in the N terminal region were analysed for their relative abilities to bind RPA, activate or repress transcription, and suppress growth of p53 null SaOs2 and H1299 cells. Both mutants of p53 with decreased RPA binding suppressed cell growth as well as wild-type p53, suggesting that p53 can suppress growth without interacting with RPA. The allele that lost most of the transcription activation function also lost most of its transcription repression activity suggesting that interaction with the same basal transcription factors are involved in both functions. This same allele bound RPA well but was defective in growth suppression. Therefore, transcription activation and/or repression appear to be more important for the growth suppression function of p53 than RPA binding.
Insights
The tumor suppressor protein p53 has multiple functions, including transcription regulation and interaction with DNA replication factor RPA. Its growth suppression activity is more dependent on transcription regulation than RPA binding.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Function
Background:
- The tumor suppressor protein p53 regulates gene transcription and interacts with the human DNA replication factor RPA.
- These activities are primarily mediated by the N-terminal 70 amino acids of p53.
- Specific sub-domains within p53 synergize for robust RPA binding.
Purpose of the Study:
- To investigate the relationship between p53's RPA binding, transcription modulation, and cell growth suppression activities.
- To identify key residues and domains involved in p53-RPA interactions.
- To determine the relative importance of RPA binding versus transcription regulation in p53's tumor suppressor function.
Main Methods:
- Analysis of p53 mutants with point mutations in the N-terminal region.
- Assays to measure RPA binding affinity.
- Experiments to assess transcription activation and repression.
- Cell growth suppression assays in p53-null cell lines (SaOs2 and H1299).
Main Results:
- Mutants with reduced RPA binding retained significant cell growth suppression activity, indicating p53 can suppress growth independently of RPA.
- A specific p53 allele that lost transcription activation also lost transcription repression, suggesting shared transcription factor involvement.
- This same allele bound RPA effectively but was deficient in growth suppression.
- Aromatic amino acids flanked by negatively charged residues are important for RPA binding.
Conclusions:
- p53's tumor suppressor function, specifically cell growth suppression, is more critically dependent on its transcription activation and repression activities than its interaction with RPA.
- The N-terminal region of p53 is crucial for its diverse functions, including RPA binding and transcription modulation.
- Understanding these distinct functional domains provides insights into p53's role in cancer.