Nucleoprotein Structures at Telomeres and Sites of DNA Damage

描述（由申请人提供）：结构性的见解可以塑造新的思维，并有助于范式转变的影响。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DNA修复途径的信号可能取决于端粒的物理变化。这个实验室以前的工作导致了端粒环（t-loops）和小端粒DNA环（t-circles）的发现，这些环现在已经从酵母菌到人类中发现，并可能有助于端粒的维持。其他人发现的一个新的参与者是与端粒结合的端粒RNA。hnRNPA 1蛋白紧密结合这种RNA，本实验室最近的工作表明，hnRNPA 1将解开双链哺乳动物端粒DNA。该研究计划采用生物化学和透射电子显微镜（EM）的独特组合。新的EM标记方法，确定蛋白质的多蛋白质复合物已经开发出来，并将沿着与新的超温和的制备方法，冷冻EM和单颗粒重建方法，融合生化检测。在AIM I中，将高度纯化的核心端粒结合蛋白（TRF 1、TRF 2、Pot 1、TPP 1、hRap 1和Tin 2）与这些蛋白在体内组装的共复合物一起组装到模型端粒模板上，检查它们的结构，并测定它们重塑端粒DNA的能力。在AIM II中，使用转基因小鼠的实验将进一步探索TRF 2的作用，并对Rad 51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hnRNPA 1在端粒上形成支架的能力，其他核心端粒结合因子可以在其上组装。hnRNPA 1在促进端粒的成环和复制性延伸中的作用将被研究。目的IV继续与Tomaska小组的长期合作，并发现了一种新的新酵母物种，具有长端粒和与人类TRF 1/2高度同源的端粒结合蛋白。这种酵母应该为人类端粒生物学提供更好的模型。上一个资助期的高生产率为未来的成功提供了强有力的衡量标准，这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这项技术应用于端粒/修复工作，许多其他实验室依赖于这些结构研究的投入。. 公共卫生相关性：端粒提供了对抗癌症的第一道防线，也提供了一个分子时钟，这是人类细胞衰老的核心。端粒结构的许多基本问题以及这些分子机器如何向DNA修复途径发出信号仍然是未知的。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。

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.