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：
8460104
负责人：
JACK D GRIFFITH
金额：
$ 30.83万
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 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460104
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. .

描述（由申请人提供）：结构见解可以塑造新的思维并贡献范式移动影响。端粒是癌症和衰老的核心。结构问题是端粒功能的核心，其中对DNA修复途径的信号可能取决于端粒的物理变化。该实验室的先前工作导致发现了端粒环（T环）和小型端粒DNA圆圈（T-Circles），这些圆圈（T-Cirdles）现已发现从酵母到人类，并可能有助于端粒维持。他人发现的一个新玩家是端粒RNA绑定在端粒上的。 HNRNPA1蛋白紧密结合了此RNA，该实验室的最新工作表明HNRNPA1将解开双链哺乳动物端粒DNA。该研究计划采用了生物化学和透射电子显微镜（EM）的独特组合。已经开发了新的EM标记方法，这些方法已开发出多蛋白质复合物中的蛋白质，并将与新的超循环制备方法，冷冻和单个颗粒重建方法一起使用，并与生化测定融合在一起。在AIM I中，高度纯化的核心端粒结合蛋白（TRF1，TRF2，POT1，TPP1，HRAP1和TIN2），并与这些蛋白质组装在一起的这些蛋白的共复合物将组装在体内组装的这些蛋白质，将被组装到模型端粒模板上，它们的结构，它们的结构，它们的结构，并重塑了远程DNA确定的DNA。在AIM II中，使用转基因小鼠的实验将进一步探测TRF2的作用，并在RAD51旁系同源物上进行工作，并将进行WRN解旋酶，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将重点放在端粒RNA和HNRNPA1上，以在其他核心端粒结合因子可能组装的端粒形成脚手架的能力。 HNRNPA1在促进端粒的循环和复制扩展中的作用将得到研究。 AIM IV继续与Tomaska集团进行了长期的合作，并发现了一种新型的新酵母菌，具有长长的端粒和与人TRF1/2同源的端粒结合蛋白。该酵母应为人类端粒生物学提供更好的模型。过去资金期的高生产率为未来的成功提供了强大的指标，这受到了强大的合作的支持。这些研究的影响很大，因为没有其他实验室将该技术应用于端粒/维修工作，并且许多其他实验室取决于这些结构研究的投入。。