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Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid
1Graduate Program In Molecular and Cellular Biology, State University of New York, Stony Brook 11794.
Abstract:
The tumor suppressor p53 protein binds to the products of several viral oncogenes, including SV40 large T antigen. We reconstructed the p53-T antigen interaction in the yeast two-hybrid system, a genetic assay that uses the reconstitution of the activity of a transcriptional activator to detect protein-protein interactions. Using mutants of T antigen known to be defective in binding to p53, we demonstrate that the two-hybrid system is more sensitive than immunoprecipitation in the detection of weak interactions. We mutagenized the murine p53 gene and screened in the yeast assay for decreased reporter gene expression indicative of the failure of p53 to bind T antigen. This screen identified 34 p53 mutants, almost all of which contain at least one mutation in the conserved domains frequently found mutated in human cancers. These results support the idea that the function of the wild-type p53 protein requires residues involved in binding to T antigen, and indicate that this approach may be generally applicable in the analysis of protein-protein interactions.
Insights
The yeast two-hybrid system detects weak protein interactions, like p53 and SV40 T antigen. This study identified 34 p53 mutants, highlighting the importance of T antigen-binding residues for p53 function.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The p53 tumor suppressor protein interacts with viral oncoproteins, such as SV40 large T antigen.
- Understanding these interactions is crucial for deciphering p53's role in cancer and viral pathogenesis.
Purpose of the Study:
- To establish and validate the yeast two-hybrid system for detecting p53-T antigen interactions.
- To identify p53 mutants defective in T antigen binding using a high-throughput yeast screen.
Main Methods:
- Reconstruction of the p53-T antigen interaction in the yeast two-hybrid system.
- Utilizing known T antigen mutants to assess the sensitivity of the yeast assay compared to immunoprecipitation.
- Mutagenesis of the murine p53 gene and screening for loss-of-function mutants in the yeast assay.
Main Results:
- The yeast two-hybrid system demonstrated higher sensitivity than immunoprecipitation for detecting weak protein interactions.
- A screen identified 34 p53 mutants exhibiting reduced reporter gene expression, indicating impaired T antigen binding.
- Most identified p53 mutants contained mutations within conserved domains frequently altered in human cancers.
Conclusions:
- The functional integrity of wild-type p53 protein relies on residues critical for T antigen binding.
- The yeast two-hybrid system offers a sensitive and broadly applicable method for analyzing protein-protein interactions.