Structural and Functional Characterization of Telomere Protection and Maintenance

端粒保护和维持的结构和功能表征

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：真核生物中线性染色体的演变对染色体的自然末端面临着几个挑战，例如错误识别为DNA损伤位点，DNA降解和不完整的DNA复制。解决这些挑战的一种解决方案是形成了一个保护性帽结构，该结构完成了远程重复序列和相关蛋白质的串联阵列。庇护所综合体可保护端粒免受DNA损伤修复（DDR）机械的处理。然而，庇护素如何重塑远程DNA并将端粒末端与受损的DNA末端区分开的分子基础尚不清楚。这些问题可以通过解决避难所结合的远程DNA的结构，并研究远程终端的Shelterin和DDR信号之间的动态相互作用。端粒区域在每个细胞分裂上缩短直到到达其结构性故障触发复制感应的极限。这就是端粒被称为内部生物时钟，这些时钟决定了体细胞的增殖寿命。在种系和干细胞中，远程酶酶的损失可以逆转，遥测酶是一种特殊的逆转录酶，可在染色体末端增加远程重复。将端粒酶募集到远程终端及其重复的加法性过程受到庇护所的严格调节。目前，我们缺乏对远程酶的机械化学周期以及庇护素组件在调节远程酶过程中具有的特定作用的综合理解。通过实时监测遥测酶重复合成将获得进一步的见解通过单个核丁基分辨率通过端粒酶。该提案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。我们有三个具体的目标：首先，我们将重建人类庇护素复合物，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EM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，我们将在流量室中的模型远程DNA底物上组装庇护素，并研究庇护素如何使用单分子特品格将端粒重塑其受保护的形式。避难所和端粒靶向蛋白之间的动态相互作用将在远程末端进行监测。我们旨在解决庇护素如何保护远程终端免受DDR信号蛋白RPA（复制蛋白A）的结合的概念困境，同时允许募集遥测酶。第三，使用光学诱捕，我们旨在确定与端粒酶相关的机械化学转变，并为通过庇护素亚基提高端粒酶加工性提高机械解释。