Analysis of the Molecular Mechanisms of Telomerase Recruitment to Telomeres and Telomerase Catalysis

摘要/摘要 人类染色体在端粒中结束，端粒是由保护物结合的重复DNA序列 蛋白质复合体(1)。在半保守的DNA复制过程中，染色体的末端是 不能复制，导致连续的染色体缩短。一旦端粒达到临界状态 长度越长，细胞进入衰老或发生凋亡(2)。为了抵消染色体缩短的影响， 持续分裂的细胞，如生殖细胞、干细胞和大多数癌细胞，都表达端粒酶， 一种含有RNA的逆转录酶(3)。端粒酶是一种独特的酶，它不断地增加 端粒重复序列，从其RNA组分复制到单链DNA悬垂上 染色体末端(4)。控制端粒酶活性的分子机制很差。 已定义，但对理解端粒维持至关重要。 保护素复合体在端粒上执行两个关键功能；它阻止端粒 它被认为是DNA损伤的部位，并将端粒酶招募到端粒(5，6)。端粒酶 招募到端粒是一个受到严格控制的过程。端粒酶存在于Cajal小体中，特化 核内的RNA处理室，贯穿细胞周期的大部分时间。在S阶段， 端粒酶被招募到端粒以维持端粒的长度(7)。虽然蛋白质-蛋白质 端粒酶与端粒结合所需的相互作用是众所周知的，空间- 端粒酶招募的时间控制是不明确的(7)。潜在的监管机制 端粒酶重新启动包括端粒酶组成的改变和掩蔽素复合体或 其组件的翻译后修饰。 端粒维持在多种人类疾病中起着重要作用。的不足之处 端粒酶组装、活性或重新聚集到端粒导致先天性角化不良，肺性 纤维化和再生障碍性贫血，以干细胞衰竭为特征的严重人类状况(8)。在……里面 此外，90%的癌症依靠端粒酶活性来实现无限期分裂(9)。因此， 了解端粒酶重新聚集到端粒和端粒酶催化的基本生物学 导致了调整这一过程的新方法，作为几种人类的治疗方法 疾病。我建议分析端粒酶重新启动的分子机制 端粒和端粒酶催化利用基因组编辑和细胞生物学相结合， 蛋白质组学、生物化学和单分子方法。我尤其会： 1.确定推动S端粒酶重新聚集到端粒的分子机制-- 阶段。使用表达标记的端粒酶和掩蔽素成分的基因组编辑的细胞系，我将 进行端粒酶向端粒转运的活细胞成像，分析端粒酶的组装状态 用细胞生物学和蛋白质组学研究细胞周期中的端粒酶和谢尔特林复合体 方法，并确定调节端粒酶运输的激酶。 2.定义端粒酶的生化和生物物理性质。使用单分子 方法，我将评估端粒酶的寡聚体状态，即控制其 内在的加工性，以及TPP1与端粒酶的相互作用对其催化循环的影响。 拟议AIMS的K99阶段将在Tom Cech博士的指导下进行， 世卫组织在培训博士后研究员方面有着非凡的记录，有30多名前导师 在世界各地著名的研究机构担任教职。切赫实验室是一家成熟的 端粒酶的生化和结构分析。再加上我在细胞方面的专业知识 基于生物学和显微镜的方法，切赫实验室提供了一个理想的实施环境 大多数拟议的研究。为了进行蛋白质组学分析，我将 与娜塔莉·安博士的实验室合作，她是使用质谱学研究的领先研究员 蛋白质翻译后修饰。安博士的专业知识和蛋白质组学核心设施 生物前沿研究所将让我在使用质谱学作为核心方面奠定坚实的基础 发现工具，一个重要的学习体验，将促进我的短期目标和我的未来 独立的事业。 我在K99阶段的目标是启动提案的目标1和2，并建立一个强大的 为成为美国一家研究机构的独立调查员奠定了基础。我的 长期目标是运行一项研究计划，专注于确保 染色体完整性，是一种在大量人类疾病中存在缺陷的过程，使用多个 学科方法包括细胞生物学、生化、生物物理学、蛋白质组学和遗传学 方法：研究方法。K99助学金将极大地帮助我提供批判性培训，帮助我获得一名教员 这个职位，让我可以开始我的独立研究生涯。

Summary/Abstract Human chromosomes end in telomeres, repetitive DNA sequences that are bound by the Shelterin protein complex (1). During semi-conservative DNA replication the extreme ends of a chromosome are unable to be duplicated, leading to successive chromosome shortening. Once telomeres reach a critical length, cells enter senescence or undergo apoptosis (2). To counteract chromosome shortening, continuously dividing cells, such as germ cells, stem cells, and most cancer cells, express telomerase, an RNA-containing reverse transcriptase (3). Telomerase is a unique enzyme that processively adds telomeric repeats, copied from its RNA component, to the single-stranded DNA overhang of chromosome ends (4). The molecular mechanisms that govern telomerase processivity are poorly defined, but are critical to understand telomere maintenance. The Shelterin complex carries out two key functions at telomeres; it prevents telomeres from being recognized as sites of DNA damage, and it recruits telomerase to telomeres (5,6). Telomerase recruitment to telomeres is a tightly regulated process. Telomerase resides in Cajal bodies, specialized RNA-processing compartments in the nucleus, throughout most of the cell cycle. During S-phase, telomerase is recruited to telomeres to maintain telomere length (7). Although the protein-protein interactions required for telomerase to associate with telomeres are well understood, the spatio- temporal control of telomerase recruitment is poorly defined (7). Potential mechanisms for regulating telomerase recruitment include alterations in composition of telomerase and the shelterin complex or post-translational modification of its components. Telomere maintenance plays an important role in multiple human diseases. Deficiencies in telomerase assembly, activity, or recruitment to telomeres cause dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, severe human conditions characterized by stem cell failure (8). In addition, 90% of cancers rely on telomerase activity to allow them to divide indefinitely (9). Therefore, understanding the basic biology of telomerase recruitment to telomeres and telomerase catalysis could lead to novel approaches to modulate this process as a therapeutic approach for several human diseases. I propose to analyze the molecular mechanisms underlying telomerase recruitment to telomeres and telomerase catalysis using genome editing and a combination of cell biological, proteomic, biochemical, and single-molecule approaches. In particular I will: 1. Determine the molecular mechanisms that drive telomerase recruitment to telomeres in S- Phase. Using genome-edited cell lines expressing tagged telomerase and shelterin components, I will conduct live cell imaging of telomerase trafficking to telomeres, analyze the assembly state of telomerase and the shelterin complex throughout the cell cycle using cell biological and proteomic approaches, and identify kinases that modulate telomerase trafficking. 2. Define the biochemical and biophysical properties of telomerase. Using single molecule approaches, I will assess the oligomeric state of telomerase, the biophysical properties that control its intrinsic processivity, and the impact of the interaction of TPP1 with telomerase on its catalytic cycle. The K99 phase of the proposed aims will be conducted under the mentorship of Dr. Tom Cech, who has an extraordinary track record in training post-doctoral fellows, with over 30 former mentees in faculty positions at prestigious research institutions worldwide. The Cech lab is an established leader in the biochemical and structural analysis of telomerase. In combination with my strong expertise in cell biological and microscopy-based approaches, the Cech lab provides an ideal environment to carry out the majority of the proposed research. For the proteomic analysis of shelterin assembly I will collaborate with the lab of Dr. Natalie Ahn, a leading researcher in using mass spectrometry to study protein post-translational modifications. Dr. Ahn's expertise and the proteomics core facility at the BioFrontiers Institute will allow me develop a strong foundation in using mass-spectrometry as a core discovery tool, a critical learning experience that will facilitate my short term goals and my future independent career. My goal for the K99 phase is to initiate Aims 1 and 2 of the proposal and build a strong foundation for the transition to becoming an independent investigator at a US research institution. My long term goal is to run a research program focused on the molecular mechanisms that ensure chromosomal integrity, a process defective in a large number of human diseases, using multi- disciplinary approaches including cell biological, biochemical, biophysical, proteomic, and genetic methods. A K99 grant would greatly aid me by providing critical training, helping me secure a faculty position, and allowing me to jumpstart my career as independent researcher.