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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要端粒是真核线性染色体的必需特征，用于区分染色体 DNA 损伤造成的末端，由双链和单链 DNA 的长重复序列组成。 Shelterin 负责保护端粒末端免受 DNA 损伤反应 (DDR) 途径的影响。对细胞健康至关重要，功能缺陷与过早衰老、遗传性疾病和癌症有关。尽管庇护蛋白在基因组维护中发挥着重要作用，但人们对其机制知之甚少。 Shelterin 由六种不同的蛋白质组成，它们以分层方式组装并它需要大多数端粒末端保护成分，并且个体敲除是有效的。 Shelterin 通常是致命的，它会将端粒末端重塑为“T 环”结构。已被确定具有 DNA 重塑能力，这是庇护林实现这一目标的分子基础阐明庇护蛋白功能的关键要求之一以及这些研究的总体目标，关键在于确定庇护蛋白结构特征的细节并检查庇护蛋白的分子相互作用拟议的研究将利用涉及 DNA 的跨学科方法来实现这一目标。生物化学、计算模型和单粒子电磁。到目前为止，在我在加州大学伯克利分校诺加利斯实验室的博士后生涯中，我已经获得了高级培训分辨率冷冻电镜结构测定称为微管的螺旋丝。展望未来，我计划专注于利用单粒子研究Shelterin在结合DNA和介导端粒末端保护中的作用为了实现这些目标，我建议：（1）确定庇护蛋白的结构。使用负染色电镜，(2) 使用冷冻电镜确定单链 DNA 保护机制，以及 (3)利用冷冻电镜检查Shelterin DNA重塑能力的分子基础。在K99培训期间，我将应用生化工具优化EM重组庇护蛋白成像，我将使用单粒子电磁方法首次可视化庇护蛋白和我将在 R00 时期使用这些信息来构建我所掌握的信息。通过研究庇护蛋白的组成变异性及其如何影响庇护蛋白 I 的结构和功能来了解。相信 Eva Nogales 和 Ahmet Yildiz 的指导和强大背景以及培训 K99/R00 奖项提供的支持将为我奠定坚实的基础，使我能够成为一名成功的独立研究者，同时阐明了庇护蛋白功能的分子机制。拟议的研究将阐明 Shelterin 结合端粒 DNA 的分子机制。可以进行功能测试的新假设，并了解庇护蛋白-DNA 相互作用的方式有助于形成可用于未来治疗的端粒末端结构。