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

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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.