Molecular Mechanisms of Telomere Length Homeostasis

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，功能失调的端粒会引发基因组不稳定、细胞衰老和机体衰老。端粒结构是通过庇护蛋白与端粒DNA重复序列结合形成核蛋白复合体来提供的。与人类的庇护蛋白类似，在裂糖酵母（Schizosaccharomyces pombe）中，庇护蛋白分别由端粒序列特异性的双链和单链DNA结合蛋白Taz1和Pot1组成，并伴随着它们的蛋白相互作用伙伴Rap1、Poz1和Tpz1，形成了连接Taz1和Pot1的“蛋白桥”。庇护蛋白保护端粒完整性并调节端粒酶。在亲本的支持下，我们通过生化方法鉴定了Tpz1突变体，这些突变体可以单独但特异性地破坏其与Poz1、Ccq1或Pot1的相互作用。利用这些Tpz1的功能分离突变体，我们发现端粒dsDNA和庇护蛋白复合物内ssDNA结合蛋白之间的完整链接是定义端粒端粒酶不可延伸状态所必需的。此外，上位分析表明Tpz1也参与了端粒向可扩展状态的激活

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.