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(申请人提供)：端粒完整性对细胞存活和增殖至关重要。因此，功能失调的端粒可引发基因组不稳定、细胞衰老和生物衰老。端粒结构是通过与端粒DNA重复序列结合形成核蛋白复合体来提供的。在裂殖型裂殖酵母中，谢尔特林是由端粒序列特异的双链和单链DNA结合蛋白Taz1和POT1分别与它们的蛋白质相互作用伙伴Rap1、Poz1和Tpz1组成的，形成了连接Taz1和POT1的蛋白质桥梁。保护素既能保护端粒的完整性，又能调节端粒酶。在父母的资助下，我们通过生化方法确定了Tpz1突变体，这些突变体可以单独但特异地干扰其与Poz1、Ccq1或POT1的相互作用。利用Tpz1的这些功能分离突变体，我们发现端粒dsDNA和Shelterin复合体中的ssDNA结合蛋白之间的完全连接是定义端粒的端粒酶不可延伸状态所必需的。此外，上位性分析表明，Tpz1也通过其自身参与了端粒到可伸展状态的激活。 与Ccq1的相互作用。我们的结果表明，Tpz1在端粒开/关二元开关中起着关键的调节作用。在此，我们请求补充资金，以利用新兴技术和跨学科团队，进一步将我们对避难所的机械性理解提升到一个新的水平。本研究的两个目标是：(1)利用化学交联-质谱分析(CXMS)技术，识别端粒内相互作用和蛋白质组分的翻译后修饰；(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Tpz1在协调端粒成分在端粒酶介导和基于重组的端粒延长中的功能作用。