The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fu- sion events. Telomerase catalyzes processive extension of telomere DNA via a unique catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase (TERT), and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural RNA and protein domains coordinate the processes of telomerase RNP assembly, cataly- sis, and recruitment to telomeres. To address this goal, we will utilize a multi-faceted experimental strategy that combines single-molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. We will study human telomerase and the enzyme from the model system Tetrahymena thermophila.
In aim 1, we will employ biochemical structure probing, single-molecule Frster resonance energy transfer (smFRET), and x-ray crystallography to analyze structural intermediate states adopted by telomerase RNA and TERT during the RNP assembly pathway.
In aim 2, we will use smFRET and a novel telomerase ac- tivity detection method to investigate conformational dynamics of protein and RNA domains that drive telomer- ase function.
In aim 3, we will study the dynamic DNA handling properties of the telomerase enzyme. These experiments will focus on the molecular mechanisms for telomerase recruitment to telomeres as well as under- standing how the intrinsic folding properties of telomere DNA regulate telomerase catalysis.
Telomerase is an essential enzyme required for maintaining the protective capping structures found at chromosome ends called telomeres. Telomerase defects arise in the majority (~90%) of human cancers and several premature aging syndromes. This proposal aims to illuminate the structural and dynamic properties of telomerase that underlie its unique cellular activity, with the long term goal of establishing a conceptual framework for developing novel approaches for di- agnosing and treating telomerase-associated diseases.
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