Towards an understanding of telomere end protection: Cryo-EM studies of shelterin structure and function

了解端粒末端保护：Shelterin 结构和功能的冷冻电镜研究

• 批准号: 9371709
• 负责人: Elizabeth Kellogg
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9371709
• 关键词: ATM Signaling Pathway; ATM activation; Affect; Architecture; Award; Binding; Biochemistry; Cell Aging; Characteristics; Chromosome Structures; Chromosomes; Complex; Computer Simulation; Coupled; DNA; DNA Binding; DNA Damage; DNA Structure; Defect; Development; Disease; Electron Microscopy; Embryo; Exhibits; Filament; Focus Groups; Foundations; Future; Gene Rearrangement; Genes; Genome; Goals; Health; Hereditary Disease; Human; Image; In Vitro; Individual; Interdisciplinary Study; Knock-out; Knowledge; Lead; Light; Link; Macromolecular Complexes; Maintenance; Malignant Neoplasms; Mediating; Mentors; Mentorship; Microtubules; Missense Mutation; Modeling; Molecular; Mus; Negative Staining; Outcome; Pathway interactions; Phosphotransferases; Play; Predisposition; Premature aging syndrome; Protein Biochemistry; Proteins; Recombinants; Recruitment Activity; Research; Research Personnel; Resolution; Resources; Rest; Role; Sampling; Single-Stranded DNA; Stretching; Structure; TERF1 gene; TINF2 gene; Testing; Therapeutic; Time; Training; Training Support; Work; base; biochemical tools; cancer cell; career; ds-DNA; experience; in vivo; insight; interdisciplinary approach; nanometer; overexpression; particle; premature; reconstitution; replication factor A; response; skills; success; telomere

Project Summary/Abstract Telomeres are a required feature of eukaryotic linear chromosomes that serve to distinguish chromosome ends from DNA damage, and consist of long repeating sequences of double-stranded and single-stranded DNA. Shelterin is responsible for protecting telomere ends from the DNA-damage response (DDR) pathway. Shelterin is crucial to cellular health, and functional defects are linked to premature aging, genetic disorders, and cancer. Despite shelterin’s important roles in genome maintenance, little is known about the mechanism by which it protects telomeres. Shelterin is composed of six different proteins, which assemble in a hierarchical manner and robustly interact in vitro. It requires most components for telomere end protection, and individual knock-outs are typically lethal. Shelterin is remodels telomere ends into a ‘t-loop’ structure. While components of shelterin have been pinpointed as having DNA-remodeling capabilities, the molecular basis of how shelterin accomplishes this is enigmatic. One of the key requirements to elucidating shelterin’s function, and the overall goal of these studies, rests in determining the details of shelterin’s structural features and to examine shelterin’s molecular interactions with DNA. The proposed research will achieve this goal using an interdisciplinary approach involving biochemistry, computational modeling, and single-particle EM. Thus far in my postdoctoral career in the Nogales lab at UC Berkeley, I have obtained training in high- resolution cryo-EM structure determination of helical filaments known as microtubules. Moving forward, I plan to focus on studying the role of shelterin in binding DNA and mediating telomere end protection using single-particle negative stain EM and cryo-EM. To achieve these goals, I propose to: (1) Determine the architecture of shelterin using negative stain EM, (2) use cryo-EM to determine the mechanism of single-stranded DNA protection, and (3) use cryo-EM to examine the molecular basis of shelterin’s DNA remodeling abilities. During the K99 training period, I will apply biochemical tools to optimize recombinant shelterin for EM imaging and I will use single-particle EM approaches to visualize, for the first time, the structure of shelterin and the details of shelterin-DNA interactions. I will use this information in the R00 period to build upon what I’ve learned by studying the compositional variability of shelterin and how it affects shelterin structure and function. I believe that the mentorship and strong background of Eva Nogales and Ahmet Yildiz together with the training support provided by the K99/R00 award will allow me to build a strong foundation to enable my success as an independent investigator while illuminating the molecular mechanism of shelterin’s function. The results of the proposed studies will be to elucidate shelterin’s molecular mechanism in binding telomere DNA. This will lead to new hypotheses that can be tested functionally, and an understanding of how shelterin-DNA interactions contributes to telomere end structure that can be exploited for future therapeutics.

项目总结/摘要 端粒是真核线性染色体的必需特征，用于区分染色体 末端的DNA损伤，并由双链和单链DNA的长重复序列组成。 庇护素负责保护端粒末端免受DNA损伤反应（DDR）途径的影响。Shelterin 对细胞健康至关重要，功能缺陷与过早衰老、遗传疾病和癌症有关。 尽管shelterin在基因组维护中起着重要作用，但人们对其作用机制知之甚少。 保护端粒。Shelterin由六种不同的蛋白质组成，它们以分级方式组装， 在体外强烈相互作用。它需要大多数的端粒末端保护组件，和个人敲除， 通常是致命的Shelterin是将端粒末端改造成“t环”结构。虽然庇护所的组成部分有 shelterin具有DNA重塑能力，这是shelterin实现这一功能的分子基础 是个谜阐明shelterin功能的关键要求之一，以及这些研究的总体目标， 在于确定shelterin的结构特征的细节，并检查shelterin的分子相互作用 用DNA拟议的研究将实现这一目标，采用跨学科的方法， 生物化学、计算建模和单粒子EM。 到目前为止，我在加州大学伯克利分校的诺加莱斯实验室的博士后生涯中，我已经获得了高- 分辨率cryo-EM结构确定螺旋丝称为微管。下一步，我计划 重点研究shelterin结合DNA和介导端粒末端保护的作用 负染电镜和冷冻电镜。为实现这些目标，我建议：（1）确定掩蔽体系结构 （2）用冷冻电镜（cryo-EM）确定单链DNA的保护机制; (3)使用冷冻电镜检查shelterin的DNA重塑能力的分子基础。 在K99培训期间，我将应用生物化学工具优化EM的重组庇护素 成像，我将使用单粒子EM方法来可视化，第一次，庇护所的结构， shelterin-DNA相互作用的细节。我将在R 00期间使用这些信息， 通过研究shelterin的组成变异性以及它如何影响shelterin的结构和功能来学习。我 我相信，伊娃诺加莱斯和艾哈迈德耶尔德兹的指导和强大的背景，加上训练 K99/R 00奖提供的支持将使我能够建立一个强大的基础，使我能够成功地作为一个 独立研究者，同时阐明shelterin功能的分子机制。的结果 拟开展的研究将是阐明shelterin结合端粒DNA的分子机制。这将导致 新的假说，可以测试功能，并了解如何庇护DNA相互作用， 有助于端粒末端结构，可用于未来的治疗。