Mechanisms of radiation sensitivity: A role forp53 in the regulation of double-strand break repair.

摘要

The p53 tumor suppressor protein is known to regulate many effectors of survival following ionizing radiation (IR) exposure, including activation of cell-cycle checkpoints, DNA replication, and initiation of apoptosis. Initial studies demonstrated that radiation sensitivity was enhanced following introduction of wild-type p53 into H1299 p53-null human lung carcinoma cells. Further investigations with synchronized cells demonstrated that while the presence of wild-type p53 protein augmented sensitivity in G1-phase cells, S- and G2/M-phase cells were unaffected by p53 status. In addition, p53-induced radiation sensitivity occurred independently of p53-mediated transcriptional activation of target genes involved in apoptosis and cell-cycle arrest, including p21Waf1/Cip1 , suggesting that an additional function of p53 was responsible for the induction of radiation sensitivity.; Double-strand break repair (DSBR) is an important determinant of cellular survival following IR exposure as un-repaired double-strand breaks result in sensitivity. The two main DSBR pathways in mammalian cells are non-homologous end-joining (NHEJ) and homologous recombination (HR) repair. In response to DNA damage, proteins involved in both NHEJ and HR form DNA repair complexes at the sites of DNA lesions and orchestrate DSBR processing. To address the question of whether p53 interacted with DSBR complexes, recombinant p53 protein was immunoprecipitated with key proteins involved in NHEJ and HR repair processes, including DNA-PKcs and Rad51. Co-immunoprecipitation analysis revealed that the both DNA-PKcs/Ku80 and Rad51/Rad52 repair complexes were inhibited in the presence of p53. Furthermore, p53-mediated inhibition of DSBR complexes resulted in decreased DNA end-rejoining capacity.; Following IR exposure, p53 function is regulated by a series of post-translational modifications of the molecule, including phosphorylation of Ser15, which promotes DNA binding and transcriptional activation. However, we demonstrated that p53 interacts with DSBR complexes independent of Ser15 phosphorylation, suggesting that Ser15 phosphorylation may regulate dual functions of p53 in (1) transcriptional activation of target molecules, and (2) protein:protein interactions and inhibition of DSBR. In addition, kinetics of Ser15 dephosphorylation in cells expressing wild-type p53 correlated temporally with DSBR complex binding and inhibition of repair. Taken together, these data highlight a novel role for p53 in the induction of radiation sensitivity through the temporal regulation of DSBR.