Telomere maintenance as a target for drug discovery.

The observation that the enzyme telomerase is up-regulated in 80-90% of cancer cells isolated from primary human tumors but is absent in neighboring cells of healthy tissue has resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches toward its inhibition. In addition to inhibitors that target the enzymatic function of telomerase, efforts toward immunotherapy using peptides derived from its catalytic subunit hTERT and hTERT-promoter driven gene therapy have made significant advances. The increased level of telomerase in cancer cells also provides a potential platform for cancer diagnostics. Telomerase inhibition leads to disruption of a cell's ability to maintain the very ends of the chromosomes, which are called telomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target. In this Perspective, interdisciplinary efforts to realize the therapeutic potential of targeting telomere maintenance with a focus on telomerase are discussed.

New models of Tetrahymena telomerase RNA from experimentally derived constraints and modeling.

The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA (TER) provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment toward understanding its nontemplating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of the Tetrahymena telomerase reverse transcriptase (tTERT) to characterize the conformational changes of tTER telomerase assembly. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footprinting allow us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major groove of the helix-capping loop.

Structures of telomerase subunits provide functional insights.

BACKGROUND: Telomerase continues to generate substantial attention both because of its pivotal roles in cellular proliferation and aging and because of its unusual structure and mechanism. By replenishing telomeric DNA lost during the cell cycle, telomerase overcomes one of the many hurdles facing cellular immortalization. Functionally, telomerase is a reverse transcriptase, and it shares structural and mechanistic features with this class of nucleotide polymerases. Telomerase is a very unusual reverse transcriptase because it remains stably associated with its template and because it reverse transcribes multiple copies of its template onto a single primer in one reaction cycle. SCOPE OF REVIEW: Here, we review recent findings that illuminate our understanding of telomerase. Even though the specific emphasis is on structure and mechanism, we also highlight new insights into the roles of telomerase in human biology. GENERAL SIGNIFICANCE: Recent advances in the structural biology of telomerase, including high resolution structures of the catalytic subunit of a beetle telomerase and two domains of a ciliate telomerase catalytic subunit, provide new perspectives into telomerase biochemistry and reveal new puzzles.