Our cells are constantly dealing with DNA damage generated by endogenous cellular activity (e.g. DNA replication) and exogenous agents (e.g. ultraviolet and ionizing radiation (IR)). The cellular stress response to DNA damage requires strict co-ordination between cell cycle checkpoint control and DNA repair. In response to DNA double-strand breaks (DNA-dsbs), members of the phosphatidylinositol 3-kinase–related kinase family (e.g. ATM and DNA-PKcs kinases) have been shown to redundantly phosphorylate substrates including the DNA-dsb marker, gamma-H2AX, and the p53 tumour suppressor protein. The p53 protein is best known as the guardian of the genome through its transcriptional-dependent and -independent functions.
Despite a clear link between ATM-dependent phosphorylation of p53 with cell cycle checkpoint control and various modes of DNA damage repair, the intracellular biology and sub-cellular localization of p53 and specifically its phosphoforms during DNA damage induction and repair remains poorly characterized. Using G0/G1 confluent primary human diploid fibroblast cultures, this thesis shows that endogenous p53, phosphorylated at serine 15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following the induction of DNA breaks. This biologically distinct sub-pool of p53Ser15 is ATM-dependent and resistant to 26S-proteasomal degradation. p53Ser15 co-localizes and co-immunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA-dsb rejoining. Sub-nuclear microbeam irradiation studies confirm that p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATMSer1981 and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX-association and altered DNA-dsb kinetics following DNA damage.
We further hypothesized that the non-specific DNA binding activity of the p53 carboxy-terminus mediates chromatin anchoring at sites of DNA damage. YFP-p53 fusion constructs expressing carboxy-terminus deletion mutants of p53 were transfected into p53-null H1299 cells to determine the role of the carboxy-terminus in chromatin-binding pre- and post-IR, independent of transcriptional activity. Within this isogenic human cell system, we observed exogenous YFP-p53WT associated with ATMSer1981 and 53BP1 within cellular chromatin in a dynamic manner. We confirmed that these associations also occurred between endogenous WTp53 with ATMSer1981 and 53BP1 within the chromatin of primary human diploid fibroblasts. YFP-p53del1-299 fusion proteins, which lack transcriptional activity and the Ser15-residue, also associated within chromatin. Ser15-phosphorylation was found not to be essential for DNA damage-induced association of p53 with chromatin or with ATMSer1981 and 53BP1. These data suggest a unique biology for p53Ser15 phosphoforms in the initial steps of DNA damage signaling and implicates ATM-p53-53BP1 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair. And we propose a model whereby a pre-existing pool of p53 that constantly scans the genome, responds immediately to radiation-induced DNA damage by virtue of its association with chromatin through its carboxy-terminus.
The consequences for these p53-ATMSer1981-53BP1 complexes following DNA damage remains to be investigated: could residual complexes be associated with decreased DNA-dsb rejoining or error-prone repair, or could these complexes signal for cell survival or cell death? Since altered p53 function and biology is an important factor in cellular carcinogenesis and response to cancer therapy, this study provides a step towards a greater understanding of WTp53 and MTp53 biology in tumour development and therapeutic resistance, in the hopes to contribute towards predicting therapeutic response and/or improving p53-targeted therapies.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/26447 |
| Date | 07 March 2011 |
| Creators | Al Rashid, Shahnaz Tahihra |
| Contributors | Bristow, Robert |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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