Friday, April 4, 2025
4/4/2025
Structure, Accessibility and Extension of Telomeric Overhangs - Project Summary: Telomeres contain repeating GGGTTA sequences and terminate with a 50-300 nt long single-stranded overhang (ssTEL). This overhang folds into tandem G-quadruplex (GQ) structures, which stabilize these otherwise vulnerable ends and play critical roles in distinguishing them from damaged DNA. In addition, telomeres are protected and organized by Shelterin, a multi-protein complex. Due to end replication problem, telomeres gradually shorten in dividing somatic cells, which undergo senescence or apoptosis when telomeres reach a critical length. However, telomere length is maintained in most cancers by activation of telomerase, a ribonucleoprotein complex that elongates telomeres using an RNA template. Human ssTEL can form 2-12 GQs, separated by unfolded regions which are potentially accessible to nucleases, DNA damage response (DDR) activators, telomerase, and telomeric repeat containing RNA (TERRA). Therefore, telomeres must perform their critical functions while their structure, stability, and accessibility are dynamically modulated. While single molecule and ensemble techniques have been successfully employed to study interactions of “single” telomeric GQs with proteins and small molecules, only few such studies have been performed on ssTEL of physiologically relevant lengths (>50 nt) due to their complex structures and limitations of employed methods. Recently, we demonstrated the exciting potential of a new approach, based on single molecule FRET-PAINT, to quantify the impact of shelterin on telomere accessibility. We also developed a computational model to interpret the implications of the observed accessibility patterns in terms of GQ folding propensity of different regions of ssTEL, cooperativity between neighboring GQs, and folding frustration. Here, we propose using single molecule fluorescence methods, including FRET-PAINT, cryo-electron microscopy and several ensemble methods, analytical modeling and Monte Carlo simulations to gain mechanistic understanding about structure and function of physiological ssTEL and their interactions with Shelterin, telomerase, TERRA, small molecules, DDR activators, and nucleases. For example, we will determine how overhang length, Shelterin, and multimeric small molecules impact the accessibility of ssTEL to telomerase, nucleases, and DDR activators. Finally, we will investigate the impact of Shelterin and small molecules on telomerase-catalyzed telomere extension and on how the folding patterns of the newly synthesized repeats evolve over time to ensure their protection against nucleases. Upon successful completion of the proposed work, we will have a single-molecule understanding of: (1) Length dependent variation of structural and kinetic characteristics of telomeric overhangs; (2) The impact of Shelterin, TERRA, and small molecules on accessibility of ssTEL to nucleic acid probes, DDR activators, nucleases, and telomerase; (3) How relevant physiological factors affect telomerase-catalyzed telomere extension and the folding patterns of newly synthesized telomeres. These studies will provide us with a detailed experimental and computational picture of the structure and dynamics of long telomeric overhangs and their interactions with relevant physiological factors. The technical advances achieved in this work will also help establishing new single molecule approaches to study repeating sequences that are encountered different settings, including major neurological disorders.
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