Nucleoprotein Structures at Telomeres and Sites of DNA Damage

端粒和 DNA 损伤位点的核蛋白结构

基本信息

批准号：
8040729
负责人：
JACK D GRIFFITH
金额：
$ 32.54万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-26 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8040729
关键词：
Affinity Age Aging Architecture Binding Binding Proteins Binding Sites Biochemical Biochemistry Biological Assay Biology Cells Collaborations Complex Cruciform DNA Cryoelectron Microscopy DNA DNA Binding DNA Damage DNA Repair DNA Repair Pathway DNA-Binding Proteins Data Distant Divalent Cations Electron Microscopy Event Fission Yeast Fluorescence Funding Future Gel Genetic Genetic Recombination Goals Hand Homologous Gene Human In Vitro Individual Insecta Kinetics Knock-out Laboratories Learning Maintenance Malignant Neoplasms Methods Metric Modeling Molecular Molecular Chaperones Molecular Conformation Molecular Machines Monovalent Cations Multiprotein Complexes Mus Nucleoproteins Nucleotides Paper Productivity Protein Binding Proteins Publishing RNA RNA Binding RNA-Binding Proteins Research Resolution Role Shapes Signal Transduction Site Staging Structure System TERF1 gene TINF2 gene Technology Telomere Maintenance Telomere-Binding Proteins Test Result Testing Thinking Transcript Transgenic Mice Transmission Electron Microscopy Work Yeast Model System Yeasts arm design dimer helicase in vivo insight melting monomer nanoscale novel novel strategies paralogous gene particle programs promoter protein complex reconstruction repaired research study scaffold success telomere

项目摘要

DESCRIPTION (provided by applicant): Structural insights can shape new thinking and contribute paradigm-shifting impact. Telomeres are central to cancer and aging. Questions of structure are central to telomere function where signaling to the DNA repair pathway likely depends on physical changes in the telomere. Previous work from this laboratory led to the discovery of telomere looping (t-loops) and small telomeric DNA circles (t-circles) which have now been found from yeast to humans and likely contributes to telomere maintenance. A new player discovered by others is telomeric RNA bound at the telomere. hnRNPA1 protein binds this RNA tightly and recent work in this laboratory revealed that hnRNPA1 will unwind duplex mammalian telomeric DNA. This research program applies a unique combination of biochemistry and transmission electron microscopy (EM). New EM tagging methods that identify proteins in multiprotein complexes have been developed and will be applied along with new ultra-gentle preparative methods, cryoEM and single particle reconstruction methods, melded with biochemical assays. In AIM I, the core telomere binding proteins (TRF1, TRF2, Pot1, TPP1, hRap1, and Tin2) highly purified and in hand together with co-complexes of these proteins assembled in vivo will be assembled onto model telomere templates, their structure examined, and their ability to remodel telomeric DNA determined. In AIM II, experiments using transgenic mice will further probe role of TRF2, and work on the Rad51 paralogs and the WRN helicase will be conducted to examine the interaction of these repair factors with the telomere complexes on telomeric DNA. AIM III will focus on telomeric RNA and hnRNPA1 in their ability to form a scaffold at the telomere upon which other core telomere binding factors may assemble. The role of hnRNPA1 in facilitating looping and replicative extension of the telomere will be investigated. Aim IV continues a long standing collaboration with the Tomaska group and the discovery of a novel new yeast species with long telomeres and telomere binding protein highly homologous to human TRF1/2. This yeast should provide a better model for human telomere biology. The high productivity of the past funding period provides a strong metric for future success and this is bolstered by strong collaborations. These studies have very high impact since no other laboratory is applying this technology to telomere/repair work and many other laboratories depend on the input from these structural studies. . PUBLIC HEALTH RELEVANCE: Telomeres provide the first line of defense against cancer, and also provide a molecular clock that is central to ageing in human cells. Many basic questions of telomere structure and how these molecular machines signal to the DNA repair pathways remain unknown. The goal of this program is to provide answers to central questions that currently limit progress and thinking in this field.

描述（由申请人提供）：结构性见解可以塑造新思维并带来范式转变的影响。端粒对于癌症和衰老至关重要。结构问题是端粒功能的核心，其中向 DNA 修复途径发出的信号可能取决于端粒的物理变化。该实验室之前的工作发现了端粒环（t 环）和小端粒 DNA 环（t 环），现在从酵母到人类都发现了它们，可能有助于端粒的维持。其他人发现的一个新参与者是结合在端粒上的端粒RNA。 hnRNPA1 蛋白与该 RNA 紧密结合，该实验室最近的工作表明 hnRNPA1 可以解开双链哺乳动物端粒 DNA。该研究项目采用了生物化学和透射电子显微镜 (EM) 的独特组合。识别多蛋白复合物中蛋白质的新 EM 标记方法已经开发出来，并将与新的超温和制备方法、冷冻电镜和单粒子重建方法一起应用，并与生化测定相结合。在 AIM I 中，高度纯化的核心端粒结合蛋白（TRF1、TRF2、Pot1、TPP1、hRap1 和 Tin2）与体内组装的这些蛋白质的复合物一起组装到模型端粒模板上，并检查其结构，以及它们重塑端粒 DNA 的能力。在AIM II中，使用转基因小鼠的实验将进一步探讨TRF2的作用，并将对Rad51旁系同源物和WRN解旋酶进行研究，以检查这些修复因子与端粒DNA上的端粒复合物的相互作用。 AIM III 将重点关注端粒 RNA 和 hnRNPA1 在端粒上形成支架的能力，其他核心端粒结合因子可以在该支架上组装。将研究 hnRNPA1 在促进端粒循环和复制延伸中的作用。 Aim IV 继续与 Tomaska 小组长期合作，并发现了一种具有长端粒和与人类 TRF1/2 高度同源的端粒结合蛋白的新型酵母菌种。这种酵母应该为人类端粒生物学提供更好的模型。过去资助期的高生产力为未来的成功提供了强有力的衡量标准，而这又得到了强有力的合作的支持。这些研究具有非常高的影响力，因为没有其他实验室将该技术应用于端粒/修复工作，并且许多其他实验室依赖于这些结构研究的输入。。公共健康相关性：端粒提供了抵御癌症的第一道防线，并且还提供了对人类细胞衰老至关重要的分子钟。关于端粒结构以及这些分子机器如何向 DNA 修复途径发出信号的许多基本问题仍然未知。该计划的目标是为目前限制该领域进展和思考的核心问题提供答案。