Characterization of the C-terminal domain of the P53 tumor suppressor
Shamloo, Bahar (2016) Characterization of the C-terminal domain of the P53 tumor suppressor. [Thesis]
Half of all cancer cases have a mutated p53 gene. The p53 protein’s C-terminal domain plays an important role in its post-translational modification. We studied the function of this domain in endogenous p53 in HCT116 human colon cancer cell lines. We generated several C-terminal mutant (CTM) cell lines through CRISPR-Cas9 mediated genome editing. The focus of this study is three of these p53 CTM cell lines that encode 1) a truncated p53 protein (p53 Δ21 / Δ21); 2) a single amino acid deleted p53 protein (p53 ΔR379 / ΔR379); and 3) a p53 protein missing the C-terminus encoded by the last p53 exon (p53 Δ26 / Δ26). We show that cells which express the p53 ΔR379 protein (with deleted Arg379), grow slower than their wild type counterparts and cannot bind to DNA targets as efficiently as WT p53. The p53 Δ21 mutant lacks 21 amino acids at the C-terminus. This mutant p53 can accumulate after DNA damage and can bind to DNA targets efficiently; nevertheless, it shows a different transcription pattern for some genes that play important roles in ribosome biogenesis stress. The p53 Δ26 expressing mutant cell line lacks the last exon of p53 on both alleles, behaves in a very different way. The phenotype of this mutant is similar to HCT116KO cells that do not express p53. These mutant cells express truncated p53 and they grow much slower and go to senescence twice as fast as HCT116KO counterparts. This p53 mutant cannot translocate into the nucleus after DNA damage induced by Doxorubicin treatment. We assessed the expression of p53 target genes by quantitative RT PCR and found defects in the expression of these genes for all our CTM cell lines. These results indicate that the p53 C-terminal domain is very critical for the protein’s function.
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