Abstract
p53 becomes activated after DNA damage and prevents cells from getting cancer by initiating cell cycle arrest or apoptosis. However, it is not well known about how p53 responds to DNA damage. To understand the mechanism of p53 response to DNA damage and its function, we have used in situ visualization analysis whose experimental system has been developed in our laboratory. We have introduced various types of damage at restricted nuclear regions of mammalian cells by irradiation with UVA laser through a microscope lens, and investigated the dynamics of p53. We expressed GFP-fused p53 in human cells and irradiated UVA laser in the nucleus of the cell. We found that p53 accumulated rapidly at the irradiated site. We generated p53 deletion constructs, which lack N-terminus or C-terminus of p53 and it turned out that the region between 102-354 amino acids including DNA binding domain and tetramerization domain is the minimal domain of p53 that is necessary for accumulation. Whereas point mutants defective in tetramer formation accumulated as well as the wild type p53, point-mutated p53 with lower DNA binding activity did not accumulate at the site of laser irradiation. From these data, we concluded that p53 accumulation is independent of p53 tetramer formation, but dependent on the ability of DNA binding. In addition, almost all of hot spot point mutants, identified as highly frequent in cancer patients, did not accumulate. These results suggest that there may be an important relationship between the inability of p53 accumulation at DNA damage and carcinogenesis.
