Direct Visualization of DNA Dynamics During the Telomerase Catalytic Cycle Reveals the Function of a Conserved Telomerase Domain

Abstract

Telomerase is an enzyme that maintains the ends of eukaryotic chromosomes and is important to our understanding of both aging and cancer. The telomerase catalytic core contains both a conserved protein subunit known as telomerase reverse transcriptase (TERT), and a conserved RNA known as telomerase RNA (TER). Telomerase recognizes its DNA substrate by base-complementarity with a region of TER, and extends the DNA using TERT's reverse transcriptase activity. Structural, functional, and kinetic studies of telomerase have been limited by its poor expression in vivo and in heterologous systems, as well as its structural heterogeneity. For this reason, this is an ideal system to study at the single molecule level, as single molecule assays do not require large amounts of material and can parse out different sub-populations of molecules. Our lab has developed a single-molecule Forster resonance energy transfer (smFRET) assay in order to monitor structural changes in the telomerase enzyme during its activity. We have used this assay in combination with several telomerase mutants to conduct structural and mechanistic studies of telomerase function. Our results demonstrate that a conserved telomerase N-terminal domain (TEN domain) stabilizes duplex formation between telomerase RNA and its DNA substrate. This TEN domain functions by altering the equilibrium between the duplex (active) state and a previously unknown alternative (inactive) binding conformation. Furthermore, we demonstrate that mutants that stabilize the alternative conformation have severe defects in telomerase activity. The discovery of this inactive alternative state suggests that stabilizing this conformation could be a useful of target of telomerase inhibitors for future cancer treatment.