The long term objective of the project is to identify the mechanism by which radiation induces G1 arrest in cells. The key step in the induction of G1 arrest is the transcriptional activation of the p21 gene (also called WAF-1/Cip-1). The hypothesis to be tested is that radiation activation of the p21 gene occurs through the binding of appropriately phosphorylated p53 to specific sequences in the p21 promoter.
Three specific aims will be undertaken. (1) The activation of p53 following the irradiation of cells may require alterations in the phosphorylation of the p53 protein. The serine phosphorylation sites in p53 will be individually point mutated to alanine to abolish radiation dependent phosphorylation. We will determine how the loss of these p53 phosphorylation sites affects the important functional properties of the p53 protein, including DNA binding, protein stability, phosphorylation status and transcriptional activation of the p21 gene. (2) The most important target of radiation activated p53 is the p21 gene. The p21 protein is able to inhibit cdk2 kinase, a kinase whose activity is required for entry into S-phase. The hypothesis that radiation activation of the p21 gene requires the binding of appropriately phosphorylated p53 will be tested. A series of deletions in the control sequences of the p21 promoter (including p53 binding sites) will be constructed. These will be used to determine which regions of the p21 promoter are required for radiation and DNA damage stimulated transcription of the p21 gene. The binding site for factors which regulate p21 transcription, including p53, will be identified. (3) The biological properties of p53 proteins containing serine mutations will be determined by measuring their ability to induce G1. The biochemical properties of the p53 serine mutations determined in (1) and (2) will then be related to their ability to induce G1 arrest. An understanding of how cells respond to DNA damage, especially mechanisms which may help the cell survive radiation, such as the p53 response, may allow new therapeutic approaches, including the design of novel therapeutic compounds, to be applied to radiation therapy.
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