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.

项目摘要/摘要 端粒是真核线性染色体的必需特征，用于区分染色体 从DNA损伤结束，由双链和单链DNA的长重复序列组成。 庇护所负责保护端粒末端免受DNA破坏响应（DDR）途径。庇护所 对于细胞健康至关重要，功能缺陷与过早衰老，遗传疾病和癌症有关。 尽管Shelterin在基因组维持中起着重要的作用，但对其机制知之甚少 保护端粒。庇护素由六种不同的蛋白质组成，这些蛋白质以层次的方式组装， 在体外稳健相互作用。它需要大多数用于端粒端保护的组件，并且单个淘汰赛是 通常是致命的。庇护所是端粒重塑的结构。庇护所的组成部分 被指出为具有DNA造型功能，这是庇护素如何实现这一目标的分子基础 是神秘的。阐明庇护所功能的关键要求之一，以及这些研究的总体目标， 基于确定庇护所结构特征的细节并检查庇护所的分子相互作用 与DNA。拟议的研究将使用涉及的跨学科方法实现此目标 生物化学，计算建模和单粒子EM。 在我在加州大学伯克利分校的Nogales实验室的博士后职业中，我已经获得了高级培训 螺旋丝的分辨率冷冻结构被称为微管。向前迈进，我打算 专注于研究庇护素在结合DNA和使用单粒子介导端粒保护中的作用 负污渍EM和Cryo-Em。为了实现这些目标，我建议：（1）确定庇护所的建筑 使用阴性染色EM，（2）使用冷冻EM确定单链DNA保护的机制，并 （3）使用冷冻EM检查庇护素DNA重塑能力的分子基础。 在K99培训期间，我将使用生化工具来优化重组庇护素的EM 成像和我将使用单粒子EM方法来可视化庇护所和庇护所的结构 庇护素-DNA相互作用的细节。我将在R00期间使用此信息来建立我的一切 通过研究庇护素的组成变异性及其如何影响庇护所结构和功能来学习。我 相信Eva Nogales和Ahmet Yildiz的精神训练和强大的背景以及培训 K99/R00奖提供的支持将使我能够建立坚实的基础，使我成为成功的成功 独立研究者在照亮庇护素功能的分子机制时。结果 拟议的研究将是为了阐明庇护素在结合端粒DNA中的分子机制。这将带领 对于可以在功能上测试的新假设以及对庇护素-DNA相互作用的理解 有助于端粒结构，可以探索以后的治疗。