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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM095850-10
Application #
9818378
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Gaillard, Shawn R
Project Start
2010-12-15
Project End
2023-11-30
Budget Start
2019-12-01
Budget End
2020-11-30
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of California Santa Cruz
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Shastry, Shankar; Steinberg-Neifach, Olga; Lue, Neal et al. (2018) Direct observation of nucleic acid binding dynamics by the telomerase essential N-terminal domain. Nucleic Acids Res 46:3088-3102
Musgrove, Cherie; Jansson, Linnea I; Stone, Michael D (2018) New perspectives on telomerase RNA structure and function. Wiley Interdiscip Rev RNA 9:
Parks, Joseph W; Stone, Michael D (2017) Single-Molecule Studies of Telomeres and Telomerase. Annu Rev Biophys 46:357-377
Parks, Joseph W; Kappel, Kalli; Das, Rhiju et al. (2017) Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis. RNA 23:175-188
Long, Xi; Parks, Joseph W; Stone, Michael D (2016) Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid. Methods 105:16-25
Akiyama, Benjamin M; Parks, Joseph W; Stone, Michael D (2015) The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids. Nucleic Acids Res 43:5537-49
Jansson, Linnea I; Akiyama, Ben M; Ooms, Alexandra et al. (2015) Structural basis of template-boundary definition in Tetrahymena telomerase. Nat Struct Mol Biol 22:883-8
Parks, Joseph W; Stone, Michael D (2014) Coordinated DNA dynamics during the human telomerase catalytic cycle. Nat Commun 5:4146
Long, Xi; Parks, Joseph W; Bagshaw, Clive R et al. (2013) Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy. Nucleic Acids Res 41:2746-55
Akiyama, Benjamin M; Stone, Michael D (2013) Structural biology: a solution to the telomerase puzzle. Nature 496:177-8

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