Nucleoprotein Structures at Telomeres and Sites of DNA Damage

描述(由申请人提供)：结构洞察力可以塑造新的思维，并有助于改变范式的影响。端粒是癌症和衰老的中心。结构问题是端粒功能的核心，DNA修复途径的信号可能依赖于端粒的物理变化。该实验室以前的工作导致发现了端粒环状(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。其他人发现的一种新的参与者是结合在端粒上的端粒RNA。HnRNPA1蛋白与这种RNA紧密结合，本实验室最近的工作表明，hnRNPA1将解开哺乳动物的双链端粒DNA。这项研究计划应用了生物化学和透射电子显微镜(EM)的独特组合。识别多蛋白复合体中蛋白质的新EM标记方法已经被开发出来，并将与新的超温和制备方法、低温EM和单颗粒重建方法以及与生化分析相结合的方法一起应用。在AIM I中，高度纯化的核心端粒结合蛋白(TRF1、TRF2、POT1、TPP1、hRap1和Tin2)以及这些蛋白在体内组装的共同复合物将组装到模型端粒模板上，检查它们的结构，并确定它们重塑端粒DNA的能力。在AIM II中，使用转基因小鼠的实验将进一步探索TRF2的作用，并将在RAD51并列基因和WRN解旋酶上进行工作，以检测这些修复因子与端粒DNA上的端粒复合体的相互作用。AIM III将重点研究端粒RNA和hnRNPA1在端粒形成支架的能力，其他核心端粒结合因子可能在其上组装。HnRNPA1在促进端粒的环和复制延伸中的作用将被研究。目的IV继续与Tomaska小组的长期合作，发现了一种新的具有与人TRF1/2高度同源的端粒和端粒结合蛋白的新酵母物种，该酵母将为人类端粒生物学提供更好的模型。过去资助期的高生产率为未来的成功提供了一个强有力的衡量标准，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，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。。

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. .