Molecular Insights into the Structure and Function of the Telomerase Holoenzyme in Tetrahymena thermophila

摘要

Telomeres are specialized, G-rich simple-sequence repeats that cap the ends of linear chromosomes to prevent genome instability. These tandem DNA repeats are bound by sequence-specific proteins to create a protective structure that marks the chromosome end thereby preventing aberrant chromosomal recombination, resection, degradation, and fusion. Due to inherent limitations of genome replication and chromosome end processing, telomeres shorten over time leading to potential loss of genetic information if not restored or maintained. The ribonucleoprotein (RNP) telomerase functions in this regard by using an integral RNA template (TER) to synthesize single stranded telomeric repeats at the chromosome end. In vitro minimal catalytic activity can be reconstituted from the telomerase protein component TERT and TER; however, in vivo biologically active holoenzyme requires further protein components for repeat addition synthesis, enzyme recruitment, and regulation in the cell. The ciliate Tetrahymena thermophila serves as an experimentally favorable model system for the study of telomerase due high levels of constitutively active enzyme and robust molecular and genetic techniques. Furthermore, our understanding of the holoenzyme is arguably best characterized from the Tetrahymena enzyme, which consists of nine protein components and the RNA (TERT, TER, p65, p50, Teb1, Teb2, Teb3, p75, p45, and p19). Despite knowledge of the overall architecture, relationships between multiple proteins within the holoenzyme and their specific physiological roles had remained unresolved.;Using a variety of in vitro and in vivo biochemical techniques, I show that the holoenzyme component p50 functions as a central hub for enzyme assembly, connecting the RNP catalytic core to the RPA-like Teb1-Teb2-Teb3 (TEB) and p75-p45- p19 (CST) subcomplexes. To answer existing questions concerning telomerase recruitment, I employ endogenously tagged holoenzyme proteins to show that all telomerase holoenzyme subunits are subject to coordinate telomere recruitment and release dependent on the cell cycle. Using domain tagging and truncation strategies, I demonstrate that the high-affinity single-stranded telomeric DNA binding component Teb1 is necessary and sufficient for interaction between telomerase and the telomere. This work supports a model for Tetrahymena telomerase-telomere recruitment that breaks the precedent established by studies in yeast and vertebrate cells: Teb1- containing holoenzyme is recruited directly to the telomeric DNA rather than telomerase recruitment by interaction with a telomere-bound protein. Together, along with ongoing studies of the Tetrahymena TEB and CST subcomplexes, these results suggest commonalities of telomerase interaction, action, and regulation at telomeres across species.