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首页> 外文学位 >The Role of the p53 Tumour Suppressor Protein in Relation to the Sensing of Ionizing Radiation-Induced DNA Double-Strand Breaks.
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The Role of the p53 Tumour Suppressor Protein in Relation to the Sensing of Ionizing Radiation-Induced DNA Double-Strand Breaks.

机译：p53肿瘤抑制蛋白在电离辐射诱导的DNA双链断裂感测中的作用。

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摘要

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.;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-p53 WT 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-p53Delta1-299 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.;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.
机译：我们的细胞不断处理由内源性细胞活性（例如DNA复制）和外源性物质（例如紫外线和电离辐射（IR））产生的DNA损伤。细胞对DNA损伤的应激反应需要细胞周期检查点控制和DNA修复之间的严格协调。响应DNA双链断裂（DNA-dsbs），磷脂酰肌醇3-激酶相关激酶家族的成员（例如ATM和DNA-PKcs激酶）已被证明可磷酸化底物，包括DNA-dsb标记，γ -H2AX和p53肿瘤抑制蛋白。 p53蛋白通过其转录依赖性和非依赖性功能而成为基因组的守护者。我们进一步假设，p53羧基末端的非特异性DNA结合活性介导了染色质锚定在DNA损伤位点。将表达p53羧基末端缺失突变体的YFP-p53融合构建体转染到无p53的H1299细胞中，以测定羧基末端在染色质结合IR之前和之后的作用，而与转录活性无关。在这个同基因的人类细胞系统中，我们以动态的方式观察到了与细胞染色质中ATMSer1981和53BP1相关的外源性YFP-p53 WT。我们证实这些关联还发生在人类AT二倍体成纤维细胞染色质内的ATMSer1981内源WTp53与53BP1之间。 YFP-p53Delta1-299融合蛋白，缺乏转录活性和Ser15-残基，也与染色质相关。发现Ser15磷酸化对于DNA损伤诱导的p53与染色质或与ATMSer1981和53BP1的结合不是必需的。这些数据表明在DNA损伤信号转导的初始步骤中，p53Ser15磷酸形式具有独特的生物学特性，并暗示基于ATM-p53-53BP1染色质的相互作用是细胞周期检查点控制和DNA修复的媒介。并且，我们提出了一个模型，通过该模型，一个预先存在的p53池会不断扫描基因组，并通过其通过羧基末端与染色质的结合而对放射线诱导的DNA损伤立即做出反应。这些p53-ATMSer1981-53BP1的后果DNA损伤后的复合物仍有待研究：残留的复合物是否与DNA-dsb的重新结合减少或容易出错的修复相关，或者这些复合物是否表示细胞存活或细胞死亡？由于p53功能和生物学特性的改变是细胞致癌和对癌症治疗反应的重要因素，因此本研究为进一步了解WTp53和MTp53生物学在肿瘤发展和治疗耐药性方面迈出了一步，希望有助于预测治疗反应和/或改善针对p53的疗法。尽管在ATM依赖的p53磷酸化与细胞周期检查点控制和各种模式的DNA损伤修复，p53的细胞内生物学和亚细胞定位以及DNA损伤期间的磷酸形式之间存在明确联系诱导和修复的特征仍然很差。利用G0 / G1融合的原代人类二倍体成纤维细胞培养物，本研究表明在DNA断裂诱导后30分钟内，丝氨酸15磷酸化的内源性p53（p53Ser15）以离散，剂量依赖性和染色质结合的形式聚集。 p53Ser15的这个生物学上不同的子池是ATM依赖性的，并且对26S-蛋白酶体降解具有抗性。 p53Ser15与g-H2AX共定位并共免疫沉淀，其动力学类似于生化DNA-dsb重新结合的动力学。亚核微束辐射研究证实，体内p53Ser15被募集到含有γ-H2AX，ATMSer1981和DNA-PKcsThr2609的DNA损伤位点。此外，使用分别表达Ser15或Ser18磷酸突变蛋白的等基因人和鼠细胞进行的研究显示，DNA损伤后核灶形成缺陷，p21WAF诱导减少，γ-H2AX缔合减少以及DNA-dsb动力学改变。

著录项

  • 作者

    Al Rashid, Shahnaz Tahihra.;

  • 作者单位

    University of Toronto (Canada).;

  • 授予单位 University of Toronto (Canada).;
  • 学科 Biophysics Medical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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