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: 9262255
• 负责人: Ahmet Yildiz
• 金额: $ 29.05万
• 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/9262255
• 关键词: Address; Aging; Architecture; Binding; Binding Proteins; Biological Assay; Biological Clocks; Cell Aging; Cell division; Chromosomes; Clinical Trials; Closure by clamp; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Structure; 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; Physiological; 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; 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自然末端提出了一些挑战，例如误识别为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。保护蛋白复合物可保护端粒免受 DNA 损伤修复 (DDR) 机制的处理。然而，shelterin 如何重塑端粒 DNA 并将端粒末端与受损 DNA 末端区分开来的分子基础仍不清楚。这些问题可以通过解析与庇护蛋白结合的端粒 DNA 的结构并研究端粒末端庇护蛋白和 DDR 信号之间的动态相互作用来解决。 端粒束在每次细胞分裂时都会缩短，直到达到 它们的结构故障会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。在生殖系和干细胞中，端粒束的丢失可以通过端粒酶来逆转，端粒酶是一种特殊的逆转录酶，可以在染色体末端添加端粒重复序列。端粒酶向端粒末端的募集及其重复添加过程受到庇护蛋白的严格调控。目前，我们对端粒酶的机械化学循环以及庇护蛋白成分在调节端粒酶持续合成能力中的具体作用缺乏全面的了解。通过实时监测端粒重复合成将获得进一步的见解 通过端粒酶以单核苷酸分辨率进行。 该提案的主要目标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，我们将重建人类庇护蛋白复合物，并使用负染色电子显微镜 (EM) 确定 apo 和 DNA 结合复合物的结构。然后，我们将尝试使用冷冻电镜获得复合物的高分辨率结构，以揭示介导端粒保护和维持的蛋白质-核酸相互作用。其次，我们将在流动室中的模型端粒 DNA 底物上组装庇护蛋白，并研究庇护蛋白如何使用单分子 FRET 将端粒重塑为受保护的形式。将在端粒末端监测庇护蛋白和端粒末端靶向蛋白之间的动态相互作用。我们的目标是解决庇护蛋白如何保护端粒末端免受 DDR 信号蛋白 RPA（复制蛋白 A）的结合，同时允许端粒酶募集的概念困境。第三，利用光学捕获，我们的目标是识别与端粒酶端粒重复合成相关的机械化学转变，并为通过庇护蛋白亚基增强端粒酶持续合成能力提供机制解释。