Molecular Mechanisms of Telomere Length Homeostasis

端粒长度稳态的分子机制

• 批准号: 8738357
• 负责人: Feng Qiao
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8738357
• 关键词: Aging; Binding; Binding Proteins; Biological Models; Cell Aging; Cell Cycle; Cell Proliferation; Cell Survival; Cell physiology; Chemicals; Chromosomes; Code; Complex; Computational Biology; Coupled; DNA; DNA Damage; Elongation by Telomerase; Emerging Technologies; Fission Yeast; Funding; Genes; Genetic; Genetic Epistasis; Genetic Recombination; Genomic Instability; Genomics; Histone Code; Homeostasis; Human; Individual; Lead; Length; Machine Learning; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Nucleoproteins; Parents; Pattern Recognition; Post-Translational Protein Processing; Proteins; Proteomics; Publishing; Recording of previous events; Regulation; Research; Resolution; Role; SS DNA BP; Site; Structure; Technology; Telomerase; Telomere Maintenance; Testing; Therapeutic; Time; Work; base; cancer cell; crosslink; design; genetic regulatory protein; member; mutant; new technology; next generation sequencing; novel; parent grant; prevent; protein complex; protein protein interaction; public health relevance; response; single molecule; stem cell differentiation; telomere

DESCRIPTION (provided by applicant): Telomere integrity is essential for cell survival and proliferation. Accordingly, dysfunctional telomeres can initiate genomic instability, cellular senescence, and organismal aging. Telomere structure is provided by binding of shelterin to telomeric DNA repeats forming a nucleoprotein complex. Similar to the human shelterin, in fission yeast, Schizosaccharomyces pombe, shelterin is composed of telomeric sequence-specific double-stranded and single-stranded DNA binding proteins, Taz1 and Pot1, respectively, accompanied by their protein interaction partners, Rap1, Poz1, and Tpz1, which form a "protein bridge" connecting Taz1 and Pot1. Shelterin protects telomere integrity as well as regulates telomerase. With the parent grant support, we biochemically identified Tpz1 mutants that can individually but specifically disrupt its interactions with Poz1, Ccq1, or Pot1. Using these separation-of-function mutants of Tpz1, we found that the complete linkage between telomere dsDNA and ssDNA binding proteins within the shelterin complex is required for defining the telomerase-nonextendible state of telomeres. Moreover, epistasis analyses revealed that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Our results suggest critical regulatory roles of Tpz1 in the telomere ON/OFF binary switch. Here, we request supplemental funds to use emerging technologies and interdisciplinary teams to further advance our mechanistic understanding of shelterin to a new level. The two aims are: (1) Identify shelterin interactomes and post-translational modifications on shelterin components using chemical cross-linking coupled with mass spectrometry analysis (CXMS); (2) Reveal the functional roles of Tpz1 in coordinating shelterin components for telomerase-mediated and recombination-based telomere elongation via analyzing telomere sequences using single molecule, real-time (SMRT) next- generation sequencing technology.

描述（由申请人提供）：端粒完整性对细胞存活和增殖至关重要。因此，功能失调的端粒可以引发基因组不稳定性、细胞衰老和生物体衰老。端粒结构是由shelterin与端粒DNA重复序列结合形成核蛋白复合物而提供的。与人类shelterin类似，在裂殖酵母裂殖酵母中，shelterin由端粒序列特异性的双链和单链DNA结合蛋白Taz 1和Pot 1组成，分别伴随着它们的蛋白质相互作用伴侣Rap 1，Poz 1和Tpz 1，它们形成连接Taz 1和Pot 1的“蛋白质桥”。Shelterin保护端粒的完整性并调节端粒酶。在父母资助的支持下，我们从生物化学上鉴定了Tpz 1突变体，这些突变体可以单独但特异性地破坏其与Poz 1，Ccq 1或Pot 1的相互作用。使用这些分离的功能突变体的TPZ 1，我们发现，端粒的dsDNA和ssDNA结合蛋白的shelterin复杂的完整的连接是必需的定义端粒的端粒酶不可延伸的状态。此外，上位性分析显示，Tpz 1也参与了端粒的激活，通过其在端粒中的表达，使端粒处于可延伸状态。 与CQQ 1的互动我们的研究结果表明，关键的调节作用Tpz 1在端粒ON/OFF二元开关。在这里，我们要求补充资金，以使用新兴技术和跨学科团队，进一步推进我们的shelterin的机械理解到一个新的水平。这两个目标是：（1）使用化学交联结合质谱分析（CXMS）鉴定shelterin相互作用组和shelterin组分上的翻译后修饰;（2）通过使用单分子实时（SMRT）下一代测序技术分析端粒序列，揭示Tpz 1在协调shelterin组分用于端粒酶介导的和基于重组的端粒延伸中的功能作用。