Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation. Although both telomerase activity and the telomerase catalytic protein component, TbTERT, have been identified in the eukaryotic pathogen Trypanosoma brucei, the RNA molecule that dictates telomere synthesis remains unknown. Here, we identify the RNA component of Trypanosoma brucei telomerase, TbTR, and provide phylogenetic and in vivo evidence for TbTR's native folding and activity. We show that TbTR is processed through trans-splicing, and is a capped transcript that interacts and copurifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD), which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences, demonstrating that telomere synthesis in T. brucei is dependent on TbTR. We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments, thus defining TbTR domains that may have important functional implications in telomere synthesis. Identification and characterization of TbTR not only provide important insights into T. brucei telomere functions, which have been shown to play important roles in T. brucei pathogenesis, but also offer T. brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.
Telomeres are hot spots for mutagenic oxidative and methylation base damage due to their high guanine content. We used single-molecule fluorescence resonance energy transfer detection and biochemical assays to determine how different positions and types of guanine damage and mutations alter telomeric G-quadruplex structure and telomerase activity. We compared 15 modifications, including 8-oxoguanine (8oxoG), O-6methylguanine (O6mG), and all three possible point mutations (G to A, T, and C) at the 3′ three terminal guanine positions of a telomeric G-quadruplex, which is the critical access point for telomerase. We found that G-quadruplex structural instability was induced in the order C < T < A ≤ 8oxoG < O6mG, with the perturbation caused by O6mG far exceeding the perturbation caused by other base alterations. For all base modifications, the central G position was the most destabilizing among the three terminal guanines. While the structural disruption by 8oxoG and O6mG led to concomitant increases in telomerase binding and extension activity, the structural perturbation by point mutations (A, T, and C) did not, due to disrupted annealing between the telomeric overhang and the telomerase RNA template. Repositioning the same mutations away from the terminal guanines caused both G-quadruplex structural instability and elevated telomerase activity. Our findings demonstrate how a single-base modification drives structural alterations and telomere lengthening in a position-dependent manner. Furthermore, our results suggest a long-term and inheritable effect of telomeric DNA damage that can lead to telomere lengthening, which potentially contributes to oncogenesis.
Telomerase is a specialized reverse transcriptase that adds GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation. We conduct a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the nucleotide reverse transcriptase inhibitors ddITP and AZT-TP to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or therapeutic 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy.
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Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.
present work in which I investigated the folding of a bacterial riboswitch that regulates gene expression during the process of transcription. I will discuss some interesting questions that this work brought up about the mechanism by which this RNA folds into its biologically active structure, and how those questions can be addressed using spectroscopic techniques that combine electronic coupling and Förster resonance energy transfer (FRET) as probes of nucleic acid structure on different length-scales. In a new family of techniques that I call FRET-filtered spectroscopy (FFS), two closely spaced fluorophores (such as fluorescent base analogues) are positioned at a location within the RNA whose local conformation is of interest. Electronic coupling between these fluorophores gives rise to changes in absorbance, circular dichroism and other spectra that can be interpreted quantitatively in terms of their Å ngstrom length-scale distance and relative orientation. An additional fluorophore, chosen to be a FRET acceptor for the coupled dimer, is placed at a location in the RNA for which nanometer length-scale fluctuations are of interest. By recording any type of excitation spectrum of the coupled dimer while detecting fluorescence either directly from it or from the acceptor, the spectra obtained can be decomposed into the individual spectra of subpopulations with different FRET efficiencies from one another. I will present results on two nucleic acid systems, one of which is designed as a spectroscopic model system with well-defined FRET efficiencies and electronic coupling strengths, and the other of which is designed to probe local and global conformational changes in the riboswitch. 195-PlatTetherless, Precise and Extended Observation of Single-Molecule FRET in an Anti-Brownian Trap A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without perturbation or constraints. Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale dynamics, but existing modalities have limitations. Solution based confocal measurements are restricted by the short (1ms), diffusion limited observation time. Surface immobilized measurements can extend the observation window, but at the expense of the molecule's translational and rotational degrees of freedom. Moreover, potential perturbations to the biomolecule's function introduced by immobilization or encapsulation are always a concern. We overcome these limitations by combining smFRET optics with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon recording of smFRET trajectories over tens of seconds in solution, without tethering the molecule to a surface. We first demonstrate ABEL-FRET using short (10-30bp) DNA rulers and achieve near shot-noise limited precision of DE0.01 for 5,000 photons. This is 10 times better than burst-based confocal measurements and ...
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