Effect of Tumor Suppressor p53 Mutations on Binding to the Mdm2 Promoter

P53, considered the "guardian of the genome", plays a central role in the cell as a tumor suppressor, regulating various cellular pathways such as apoptosis, DNA repair, and the cell cycle. P53 initiates various cell processes through its binding to DNA and serving as a transcription factor. P53 activity is regulated by murine double minute 2 (MDM2), which also promotes its degradation. P53 regulates MDM2 gene expression, resulting in more MDM2 and more p53 inactivation. Mutations in p53 can cause a loss of function, preventing p53 from binding to DNA and initiating a cellular response. Alternatively, they could cause a gain of function, resulting in an oncogenic protein. P53 mutations are the most common mutations found in cancer cells and are associated with many forms of cancer. The goal of this work is to examine the most common p53 mutations, their ability to bind to the mdm2 promoter, and transform the cells. WT p53 and three of its mutants (R273C, R248Q, and R175H) were overexpressed and purified from E.coli. The expression and purification were confirmed by SDS-PAGE and Western blot analysis. The mdm2 promoter was amplified using PCR. Results showed that WT and p53 mutants were expressed and purified and the mdm2 promoter of correct length was purified at large quantities. Future work includes overexpressing p53 and its mutants under new conditions to produce a higher yield of purified protein and determining their ability to bind to DNA and transform the cells.