Single-molecule studies of T-loop formation and telomerase recruitment at telomeres

Binding of the Shelterin complex to telomeres protect chromosome ends from recognition by DNA damage repair (DDR) pathways, preventing genomic instability and regulating telomerase recruitment. However, the mechanism of Shelterin activity remains unknown, in particular its role in T-loop formation and telomerase recruitment. I will reconstitute the Shelterin bound telomere in vitro and use single-molecule and structural approaches to answer three key questions: How is the Shelterin complex recruited to telomeres?; How does T-loop structure prevent recognition by the DDR?; How does Shelterin regulated telomere length through recruitment of telomerase? I will use optical-tweezers combined with confocal microscopy to allow observation of Shelterin binding, T-loop formation and telomerase recruitment in real-time at single-molecule resolution. I will develop a novel telomeric DNA forceps technique to directly probe the formation of T-loop structures and telomere extension by telomerase. I will investigate Shelterin induced DNA structural changes through force spectroscopy and atomic force microscopy. I will determine the dynamic structure of the Shelterin complex and T-loop through combined structural single-molecule FRET and molecular dynamic simulations integrated with cryo-EM structural data. Together these novel approaches will provide unprecedented mechanistic understanding of the role of Shelterin in telomere homeostasis and mechanisms of dysregulation in disease.