Structural understanding of human shelterin complex assembly at telomere and its regulation mechanism of telomerase activity

总结 端粒是位于染色体末端的重复DNA和相关蛋白质， 基因组稳定性shelterin复合物是一组由六个成员组成的蛋白质，它对端粒的长度起着重要的作用 维持，其通过调节端粒酶的募集和作用而发生。此外，本发明还提供了一种方法， shelterin还保护染色体末端不被DNA损伤修复错误识别 机制等端粒生物学紊乱是许多人类疾病的根本原因。突变 端粒酶或shelterin成分，导致端粒维持的丧失，导致先天性角化不良， 肺纤维化和再生障碍性贫血。它的医学重要性进一步表现在90%的癌症 依赖于端粒酶的过度激活来持续增殖。尽管庇护所扮演着重要的角色， 基因组的稳定性，一个一致的框架，了解其机制，在端粒的维护还没有 确立了习一个关于shelterin如何保护染色体末端并限制端粒酶进入染色体的主要模型， 端粒尾部是端粒环模型。其他更简单的模型包括DNA封端或压缩， 形成隐蔽的结构。虽然这些模型是从DNA的重塑性质， 单个的shelterin蛋白，作为一个功能性的多亚基复合体， 仍然模棱两可。shelterin复合物如何将端粒DNA组织成各种结构， 功能以及它们如何在端粒的端粒酶延伸中在抑制和允许作用之间切换 是下一个关键问题。答案在于这些过程的分子机制，因此， 本研究的目的是阐明shelterin复合物调节端粒酶功能的结构基础 端粒的募集和延长。将采用多尺度和跨学科的方法，其中包括 生物化学、生物物理学、细胞生物学和冷冻EM技术。具体而言，本提案旨在确定：（1） shelterin复合物如何将端粒DNA组织成调节端粒酶的各种结构 可及性，（2）与端粒DNA组装的shelterin复合物的结构，以及（3）shelterin 复杂的开关从端粒封端端粒酶刺激。在K99阶段，根据 Tom Cech博士的指导下，AFM成像与生化分析将用于表征高阶 不同shelterin复合物形成的DNA-蛋白质结构及其对端粒酶募集的影响 活动将被测量。在Zhiheng Yu博士的额外支持下，将获得冷冻EM技能 同时测定shelterin与端粒DNA的冷冻电镜结构。这将有助于使用 cryo-EM来研究在独立R 00期期间shelterin调节端粒酶的结构基础。 这一提议的结果将为理解庇护机制提供一个多尺度框架。 在端粒维持中的功能，也可能为开发治疗和 诊断策略，以打击人类疾病的端粒功能障碍。

Summary Telomeres are repetitive DNA and associated proteins at the ends of the chromosomes that are important for genome stability. The shelterin complex, a six-member group of proteins, is important for telomere length maintenance, which occurs through regulating the recruitment and action of the telomerase enzyme. In addition, shelterin also protects the chromosome ends from being incorrectly recognized by DNA damage repair mechanisms. Disorders in telomere biology are an underlying cause of many human diseases. Mutation of telomerase or shelterin components, resulting in loss of telomere maintenance, leads to dyskeratosis congenita, pulmonary fibrosis and aplastic anemia. Its medical importance is further shown by the fact that 90% of cancers depend on hyper-activation of telomerase for persistent proliferation. Despite the critical roles shelterin plays in genome stability, a consistent framework of understanding its mechanism in telomere maintenance has not been established. A leading model of how shelterin can protect chromosome ends and restrict telomerase access to the telomeric tail is the telomere-loop model. Other simpler models include DNA end-capping or compaction by shelterin to form reclusive structures. While these models are derived from the DNA remodeling properties of individual shelterin proteins, the collective roles shelterin proteins play as a functional multisubunit complex are still ambiguous. How a shelterin complex organizes telomeric DNA into various architectures for their regulatory functions and how they switch between inhibitory and permissive roles in telomerase elongation of telomeres are the next key questions. The answers lie in the molecular mechanisms of these processes and hence, the goal of this proposal is to elucidate the structural basis of shelterin complex functions in regulating telomerase recruitment and elongation of telomeres. A multiscale and interdisciplinary approach will be used, which includes biochemistry, biophysics, cell biology and cryo-EM techniques. Specifically, this proposal aims to determine: (1) How the shelterin complex organizes telomeric DNA into various architectures that regulate telomerase accessibility, (2) The structure of the shelterin complex assembled with telomeric DNA, and (3) How a shelterin complex switches from telomere end-capping to telomerase stimulation. During the K99 phase, under the mentorship of Dr. Tom Cech, AFM imaging with biochemical assays will be used to characterize higher-order DNA-protein architectures formed by various shelterin complexes, and their effects on telomerase recruitment and activity will be measured. With additional support from Dr. Zhiheng Yu, a cryo-EM skill set will be acquired while determining the cryo-EM structure of shelterin in association with telomeric DNA. This will facilitate using cryo-EM to study the structural basis of telomerase regulation by shelterin during the independent R00 phase. The results of this proposal would provide a multiscale framework for understanding the mechanisms of shelterin functions in telomere maintenance, and also potentially provide new avenues in developing therapeutic and diagnostic strategies to combat human diseases of telomere dysfunction.