Structural and Functional Characterization of Telomere Protection and Maintenance

端粒保护和维持的结构和功能表征

• 批准号: 9083326
• 负责人: Ahmet Yildiz
• 金额: $ 29.23万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9083326
• 关键词: Address; Aging; Architecture; Binding; Binding Proteins; Biological Assay; Biological Clocks; Cell Aging; Cell division; Chromosomes; Clinical Trials; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Repair; DNA biosynthesis; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Electron Microscopy; Elongation by Telomerase; Energy Transfer; Eukaryota; Evolution; Failure; Functional disorder; Germ Lines; Human; In Vitro; Length; Longevity; Maintenance; Malignant Neoplasms; Mechanics; Mediating; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Negative Staining; Nucleic Acids; Nucleotides; Pharmaceutical Preparations; Process; Proteins; RNA-Directed DNA Polymerase; Recombinants; Recruitment Activity; Resolution; Ribonucleoproteins; Role; Signal Transduction; Signaling Protein; Site; Slide; Somatic Cell; Stem cells; Structure; Surface; System; Telomerase; Telomere Maintenance; Testing; Time; Tissues; Up-Regulation; base; biophysical techniques; cancer therapy; imaging platform; insight; optical traps; public health relevance; reconstitution; replication factor A; single molecule; single-molecule FRET; telomere; telomere loss; temporal measurement; tool

 DESCRIPTION (provided by applicant): The evolution of linear chromosomes in eukaryotes poses several challenges for the natural ends of chromosomes, such as misrecognition as DNA damage sites, DNA degradation, and incomplete DNA replication. A solution to these challenges has been the formation of a protective cap structure comprising a tandem array of telomeric repeats and associated proteins. The shelterin complex protects telomeres against processing by the DNA damage repair (DDR) machinery. However, the molecular basis of how shelterin remodels telomeric DNA and distinguishes telomere termini from damaged DNA ends remains unclear. These questions can be addressed by resolving the structure of shelterin-bound telomeric DNA and investigating the dynamic interplay between shelterin and DDR signals at the telomeric terminus. Telomeric tracts shorten upon each cell division until reaching a limit at which their structural failure triggers replicative senescence. Thus, telomeres are referred to as internal biological clocks that determine the proliferative lifespan of somatic cell. In germ-line and stem cells, the loss of telomeric tracts can be reversed by telomerase, a special reverse transcriptase that adds telomeric repeats to chromosome ends. Recruitment of telomerase to the telomeric terminus and its repeat addition processivity are tightly regulated by shelterin. We currently lack an integrated understanding of telomerase's mechanochemical cycle and the specific roles that shelterin components have in modulating telomerase processivity. Further insights will be gained by real-time monitoring of telomeric repeat synthesis by telomerase at a single nucleotide resolution. The primary objective of this proposal is to develop structural and single-molecule biophysical approaches in vitro to dissect the molecular mechanism of telomere maintenance and protection. We have three specific aims: First, we will reconstitute the human shelterin complex and determine the structure of the apo and the DNA-bound complexes using negative stain electron microscopy (EM). We will then attempt to obtain a high resolution structure of the complex using cryo-EM to reveal the protein-nucleic acid interactions that mediate the protection and maintenance of telomeres. Second, we will assemble shelterin on a model telomeric DNA substrate in a flow chamber and study how shelterin remodels telomeres into their protected form using single-molecule FRET. The dynamic interplay between shelterin and telomere- end targeting proteins will be monitored at telomeric terminus. We aim to address the conceptual dilemma of how shelterin protects telomeric terminus against binding of a DDR signaling protein, RPA (replication protein A), while allowing the recruitment of telomerase. Third, using optical trapping, we aim to identify the mechanochemical transitions associated with telomeric repeat synthesis by telomerase and provide a mechanistic explanation for the enhancement of telomerase processivity by shelterin subunits.

 描述（由申请人提供）：真核生物中线性染色体的进化对染色体的天然末端提出了几个挑战，例如错误识别为DNA损伤位点、DNA降解和不完整的DNA复制。这些挑战的解决方案是形成包含端粒重复序列和相关蛋白质的串联阵列的保护帽结构。shelterin复合物保护端粒免受DNA损伤修复（DDR）机制的处理。然而，shelterin如何重塑端粒DNA和区分端粒末端从受损的DNA末端的分子基础仍然不清楚。这些问题可以通过解析shelterin结合的端粒DNA的结构和研究shelterin和DDR信号在端粒末端之间的动态相互作用来解决。 端粒束在每次细胞分裂时缩短， 这是它们结构失效引发复制衰老的极限。因此，端粒被称为决定体细胞增殖寿命的内部生物钟。在生殖细胞和干细胞中，端粒束的丢失可以通过端粒酶逆转，端粒酶是一种特殊的逆转录酶，可以将端粒重复序列添加到染色体末端。端粒酶的端粒末端的招聘和重复添加的持续性受到shelterin的严格调控。目前，我们缺乏一个完整的理解端粒酶的机械化学循环和特定的作用，shelterin组件在调节端粒酶的加工能力。通过对端粒重复序列合成的实时监测将获得进一步的见解 通过端粒酶在单核苷酸分辨率。 本研究的主要目的是在体外开发结构和单分子生物物理方法，以剖析端粒维持和保护的分子机制。我们有三个具体目标：首先，我们将重建人类shelterin复合物，并确定载脂蛋白和DNA结合复合物的结构，使用负染色电子显微镜（EM）。然后，我们将尝试获得一个高分辨率的结构复杂的cryo-EM揭示蛋白质-核酸的相互作用，介导的保护和维护端粒。其次，我们将组装shelterin的模型端粒DNA基板在流动室和研究如何shelterin改造端粒到其保护形式使用单分子FRET。在端粒末端监测shelterin和端粒末端靶向蛋白之间的动态相互作用。我们的目标是解决概念上的困境如何shelterin保护端粒末端结合的DDR信号蛋白，RPA（复制蛋白A），同时允许端粒酶的招聘。第三，利用光学捕获，我们的目标是确定与端粒重复合成端粒酶的机械化学转换，并提供一个机制的解释增强端粒酶的加工能力的shelterin亚基。