Abstract
Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA–DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity.
Highlights
Telomerase is a ribonucleoprotein enzyme that maintains the ends of eukaryotic chromosomes by synthesizing repetitive DNA sequences that serve as the foundation for protective nucleoprotein structures called telomeres [1]
Previous experiments investigating the role of the TEN domain established the TEN domain as an important site of DNA interaction and identified TEN domain mutants that severely affect the rate of repeat addition processivity (RAP) [8,9,11,15]
These assays revealed that telomerase bound to a DNA primer exists in two distinct conformations that are in dynamic equilibrium (Figure 2)
Summary
Telomerase is a ribonucleoprotein enzyme that maintains the ends of eukaryotic chromosomes by synthesizing repetitive DNA sequences that serve as the foundation for protective nucleoprotein structures called telomeres [1]. Inappropriate telomerase activation helps to confer the ability for cells to divide indefinitely and is associated with ∼90% of human cancers, making telomerase a promising target for potential cancer therapies [4]. TERT is tightly associated with TER, and functions by repetitively reverse transcribing a short template region of TER into telomeric DNA [5]. The template region basepairs with the DNA primer to form an RNA–DNA hybrid that is recognized by the TERT active site (Figure 1B) [6]. The telomerase catalytic cycle can be sub-divided into two distinct activities: nucleotide addition processivity (NAP) and repeat addition processivity (RAP). During RAP the nascent DNA must dissociate from the RNA template, re-anneal downstream and enter the TERT active site for the subsequent round of NAP [5] (Figure 1B)
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