Molecular Basis of Genome Organization and Integrity Using Cryo-EM

使用冷冻电镜研究基因组组织和完整性的分子基础

• 批准号: 9922323
• 负责人: Elizabeth Kellogg
• 金额: $ 24.9万
• 依托单位: CORNELL UNIVERSITY
• 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 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922323
• 关键词: ATM Signaling Pathway; ATM activation; Affect; Architecture; Award; Binding; Biochemistry; Cell Aging; Characteristics; Chromosome Structures; Chromosomes; Complex; Computer Models; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Structure; Defect; Development; Disease; Electron Microscopy; Embryo; Exhibits; Filament; Focus Groups; Foundations; Future; Gene Rearrangement; Genes; Genetic Diseases; Genome; Goals; Health; 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; 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 resolution; overexpression; particle; premature; reconstitution; recruit; 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，用来区分染色体 末端来自DNA损伤，由双链和单链DNA的长重复序列组成。 保护素负责保护端粒末端不受DNA损伤反应(DDR)途径的影响。遮蔽物 对细胞健康至关重要，而功能缺陷与早衰、遗传性疾病和癌症有关。 尽管庇护素在基因组维持中起着重要作用，但人们对它的机制知之甚少 保护端粒。谢尔特林由六种不同的蛋白质组成，它们以分层的方式组装，并 在体外进行强有力的相互作用。它需要用于端粒末端保护的大多数组件，个别敲除的是 通常是致命的。掩蔽素是将端粒末端重塑成T-环结构。而避难所的成分有 被精确定位为具有DNA重塑能力，这是避孕药实现这一功能的分子基础 令人费解。阐明庇护素功能的关键要求之一，也是这些研究的总体目标， 在于确定防护素的结构特征的细节，并检查防护素的分子相互作用 有DNA的。拟议的研究将使用跨学科的方法实现这一目标，包括 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莱斯实验室的博士后生涯中，我接受了高级- 螺旋细丝称为微管的分辨低温电子显微镜结构测定。展望未来，我计划 重点研究保护素在结合DNA和单颗粒介导端粒末端保护中的作用 阴性染色EM和冷冻-EM。为了实现这些目标，我建议：(1)确定避难所的架构 用负染色EM，(2)用冷冻-EM来确定单链DNA的保护机制，以及 (3)用冷冻-电子显微镜检测细胞外基质的DNA重塑能力的分子基础。 在K99培训期间，我将应用生化工具来优化EM的重组保护素 成像和我将第一次使用单粒子EM方法来可视化避难所的结构和 避难所蛋白与DNA相互作用的细节。我将在R00期间使用此信息来构建我已有的内容 通过研究庇护素的组成变异性以及它如何影响庇护素的结构和功能而了解到。我 相信伊娃·诺加莱斯和艾哈迈德·耶尔迪兹的导师和强大的背景一起训练 K99/R00奖项提供的支持将使我建立一个坚实的基础，使我能够作为一名 独立研究人员，阐明了庇护素作用的分子机制。评选结果 建议的研究将是阐明Shelterin结合端粒DNA的分子机制。这将导致 到可以在功能上测试的新假说，以及对保护素-DNA相互作用的理解 有助于端粒末端结构，可用于未来的治疗。