Single-molecule insights into an age old problem: determining how Shelterin safeguards telomere structure and stability

背景：端粒解决了线性染色体末端的两个问题：末端复制问题，即每个复制周期染色体末端的DNA丢失；末端保护问题，保护染色体末端防止染色体融合。端粒内稳态是至关重要的，因为端粒复合物的突变驱动癌症的发展，并由于端粒延长或缩短的失调而加速衰老。庇护蛋白是一种大型蛋白质复合物，与端粒结合，是维持端粒稳态的核心。该项目将使用我们最近开发的体外重构端粒系统来确定Shelterin如何在单分子和细胞水平上协调多个端粒活性。这种方法将首次直接观察DNA端粒结构，精确到双螺旋结构的分辨率，同时允许直接监测单个庇护蛋白复合物的活性。产生端粒底物含有g -四联体或r -环。确定不同的端粒结构如何在单分子水平上影响庇护素的活性3。探索庇护蛋白活性的差异如何影响体内t -环稳定性和末端保护：衰老是包括阿尔茨海默病和癌症在内的一些最普遍的慢性疾病的主要驱动因素。了解衰老的生理机制可以预防多种疾病。缺乏重构的端粒系统意味着基本的机制细节仍然未知。我们已经成功纯化了整个Shelterin复合物，并证明它是有活性的，能够在体外重建Shelterin介导的端粒功能。我们与领先的工业合作伙伴（Bruker, Lumicks）一起开发了针对该项目量身定制的单分子实验方法。这将有助于首次直接可视化Shelterin招募和最终保护活动，从而前所未有地深入了解Shelterin如何调节这些过程。实验方法：该项目结合了多个尖端的单分子技术，以及这些领域领导者的专业知识，直接可视化端粒过程。在这个项目中，您将接受高分辨率AFM的培训，这将使您能够将不同的端粒DNA结构可视化到双螺旋的分辨率。您将使用和开发我们的开放式定量图像分析工具，整合机器学习方法来可视化和量化Shelterin结合端粒DNA中的结构特征。您将使用我们的光学镊子装置与共聚焦显微镜在单分子分辨率下观察端粒DNA上的动态庇护蛋白活性。这些生物物理方法将得到已建立的细胞和遗传分析的支持，以验证体内的发现。最后，我们将描述癌症和衰老相关的突变，以了解它们如何失调端粒功能。

Background:Telomeres solve two problems with linear chromosome ends: the end-replication problem, DNA loss from the end of chromosomes every replication cycle; and the end-protection problem, protection of chromosome ends to prevent chromosome fusions. Telomere homeostasis is vital, as mutations in telomeric complexes drive cancer development and accelerate ageing due to dysregulated telomere lengthening or shortening. Shelterin, a large protein complex, binds to telomeres and is central to maintaining telomere homeostasis. This project will use our recently developed in vitro reconstituted telomeric system to determine how Shelterin orchestrates multiple telomere activities at the single-molecule and cellular level. This approach will for the first time directly observe telomeric DNA structure, down to the resolution of the double-helix, whilst allowing direct monitoring the activity of individual Shelterin complexes.Objectives:1. Generate telomeric substrates containing G-quadruplexes or R-loops2. Determine how different telomeric structures effect activity of Shelterin at the single-molecule level3. Probe how differences in Shelterin activity affect T-loop stability and end-protection in vivoNovelty:Ageing is a primary driver of some of the most prevalent chronic diseases including Alzheimer's and cancers. Understanding the physiological mechanisms of ageing could prevent a wide variety of diseases. A lack of a reconstituted telomeric system means fundamental mechanistic details remain unknown. We have successfully purified the entire Shelterin complex and demonstrated that it is active, enabling reconstitution of Shelterin mediated telomere function in vitro. We have developed single-molecule experimental approaches tailored to this project with leading industrial partners (Bruker, Lumicks). This will facilitate the first direct visualisation of Shelterin recruitment and end-protection activities, giving unprecedented insight into how Shelterin modulates these processes.Experimental Approach:This project combines multiple cutting-edge single-molecule techniques, and the expertise of leaders in these fields, to directly visualise telomeric processes. In this project you will receive training in high-resolution AFM which will enable you to visualise different telomeric DNA structures down to the resolution of the double-helix. You will use and develop our open quantitative image analysis tools, integrating machine learning approaches to visualise and quantify structural features within Shelterin bound telomeric DNA. You will observe dynamic Shelterin activity on telomeric DNA at single-molecule resolution using our optical-tweezers setup which is combined with confocal microscopy. These biophysical approaches will be supported by established cellular and genetic assays to validate findings in vivo. Finally, we will characterise cancer and ageing associated mutations to understand how they dysregulate telomere function.