A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Australia
- Illawara Health and Medical Research Institute, Australia
- Children’s Medical Research Institute, University of Sydney, Australia
- Department of Chemistry, McGill University, Canada
- School of Science, College of Science, Engineering and Health, RMIT University, Australia
- Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Srinakharinwirot University, Thailand
- Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Japan
- Institut Curie, PSL Research University, France
- Université Paris Sud, Université Paris-Saclay, France
- Instituto de Química Física ‘Rocasolano’, CSIC, Spain
Figures
Figure 1
Telomerase extends a parallel intramolecular G-quadruplex.
(A) Schematic of the likely topology of (top) an antiparallel G-quadruplex formed from unmodified telomeric 22-mer 22G0 and (bottom) a parallel unimolecular G-quadruplex formed from a telomeric …
Figure 2 with 4 supplements
Telomerase unfolds a parallel intramolecular G-quadruplex.
(A), (D), (G) Representative single-molecule acceptor (purple) and donor (blue) intensities of F-22G3 molecules over time (top panels), and the FRET traces (bottom panels) representing the ratio of …
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Figure 2—source data 1
source data for the graph shown in Figure 2C.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Control demonstrating G4 origin of ~0.5 FRET value of F-22G3.
Representative FRET trace (A) and heat map of the distribution of FRET intensities (B, n = 50) of F-22G3, surface immobilized in KCl-containing buffer, which was replaced with LiCl-containing buffer …
Figure 2—figure supplement 2
Histogram representations of F-22G3 FRET data.
(A–C) Histograms showing changes in FRET values of a population of F-22G3 molecules under different experimental conditions over 150 s, binned into 15–60 s time intervals. (D) Histogram showing …
Figure 2—figure supplement 3
Control for effect of dNTPs on F-22G3 FRET.
Histograms showing changes in FRET values of a population of F-22G3 molecules in the presence of dNTPs but absence of telomerase over 150 s, binned into 15–60 s time intervals. n = 44 molecules.
Figure 2—figure supplement 4
The unfolding rate of F-22G3, calculated by fitting the dwell time distributions of the intermediate FRET state (see Figure 2G) to a gamma distribution (n = 76 molecules).
N represents the number of kinetic steps in the best-fitting equation; fitting parameters (Chi-square and associated p-value) are shown in the table.
Figure 3 with 1 supplement
Telomerase extends a tetrameric parallel G-quadruplex.
(A) Schematic representation of F-[7GGT]4 used in smFRET studies. Blue star: AlexaFluor 555 (donor dye); purple star: AlexaFluor 647 (acceptor dye); red circle: biotin; yellow square: Neutravidin. (B…
Figure 3—figure supplement 1
Properties of tetrameric G4 with added FRET dyes.
(A) Circular dichroism (CD) spectra of 10 µM fluorophore-modified F-[7GGT]4 and unmodified [7GGT]4. (B) Thermal stability of modified and unmodified [7GGT]4, measured using CD at 260 nm. Since …
Figure 4 with 4 supplements
Telomerase unfolds a tetrameric parallel G-quadruplex.
(A), (D), (G) Representative single-molecule acceptor (purple) and donor (blue) intensities of F-[7GGT]4 molecules over time (top panels), and the corresponding FRET traces (bottom panels). (B), (E),…
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Figure 4—source data 1
source data for the graph shown in Figure 4C.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Histogram representations of F-[7GGT]4 data.
(A–C) Histograms showing changes in FRET values of a population of molecules under different experimental conditions over 160 s, binned into 15–85 s time intervals. (D) Histogram showing three …
Figure 4—figure supplement 2
Control demonstrating G4 origin of ~0.5 FRET value of F-[7GGT]4.
Representative FRET trace (A) and heat map of the distribution of FRET intensities (B), n = 52) of F-[7GGT]4, surface immobilized in KCl-containing buffer, which was replaced with LiCl-containing …
Figure 4—figure supplement 3
Control for effect of dNTPs on F-[7GGT]4 FRET.
Histograms showing changes in FRET values of a population of F-[7GGT]4 molecules in the presence of dNTPs but absence of telomerase over 150 s, binned into 15–85 s time intervals. n = 110 molecules.
Figure 4—figure supplement 4
Unfolding rates of F-[7GGT]4 under different experimental conditions.
(A to C) The unfolding rates were calculated by fitting the dwell time distributions of the intermediate FRET state (see center schematic, left panel) to a gamma distribution, for F-[7GGT]4 in the …
Figure 5 with 2 supplements
Partial unfolding of G4 does not require telomerase catalytic activity, and can be induced by the RNA template.
(A), (C), (F), (H) Examples of individual F-[7GGT]4 FRET traces under the indicated experimental conditions over 160 s. (B), (D), (G), (I) Heat maps of the distribution of FRET trajectories over …
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Figure 5—source data 1
Source data for the graph shown in Figure 5E.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig5-data1-v2.xlsx
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Figure 5—source data 2
Source data for the graph shown in Figure 5J.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Telomerase activity assay using purified catalytically inactive human telomerase (D712A) compared with wild type (WT) telomerase.
Upper gel shows products of telomerase extension of oligonucleotide Bio-L-18GGG (1 µM; Supplementary file 1); lower panel shows a northern blot of the amount of hTR in each telomerase preparation, …
Figure 5—figure supplement 2
Effect of RNA.10C concentration on F-[7GGT]4 FRET.
Histograms (A, B) and representative traces (C, D) of the FRET values of a population of F-[7GGT]4 molecules, in the presence of the indicated concentrations of oligonucleotide RNA.10C; n = 84 (A) …
Figure 6 with 3 supplements
Telomerase translocation leads to complete G4 unfolding.
(A), (E), (I) Schematic diagrams showing alignment of the telomerase template RNA with 22G3 DNA and template-directed incorporation of ddTTP (A), dTTP followed by ddATP (E) or dTTP, dATP and ddGTP (I…
Figure 6—figure supplement 1
Quantitation of data in Figure 6.
Plot of the percentage of molecules showing no change in FRET, a single FRET drop, or a two-step FRET drop, during telomerase extension of F-22G3 in the presence of the indicated combinations of …
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Figure 6—figure supplement 1—source data 1
Source data for the graph shown in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Telomerase unfolds intra- and intermolecular G4 using same mechanism.
(A), (D), (G) Examples of individual F-[7GGT]4 FRET trajectories in the presence of telomerase and the indicated combinations of nucleotides. (B), (E), (H) Heat maps of the distribution of FRET …
Figure 6—figure supplement 3
Quantitation of data in Figure 6—figure supplement 2.
Plot of the percentage of molecules showing no change in FRET, a single FRET drop, or a two-step FRET drop, during telomerase extension of F-[7GGT]4 in the presence of the indicated combinations of …
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Figure 6—figure supplement 3—source data 1
Source data for the graph shown in Figure 6—figure supplement 3.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig6-figsupp3-data1-v2.xlsx
Figure 7 with 5 supplements
Partial inhibition of unfolding of F-22G3 by ligands NMM, SST16 and PhenDC3.
(A – I) Representative FRET trajectories, heat maps and transition density plots of F-22G3 in the presence of NMM (A – C; 800 μM in folding reaction), SST16 (D – F; 5 μM) or PhenDC3 (G – I; 1 μM), …
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Figure 7—source data 1
Source data for the graph shown in Figure 7J.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Structures of G4-stabilizing ligands used in this study.
Figure 7—figure supplement 2
Demonstration of ligand-mediated stabilization of F-22G3.
(A – C) CD spectra and (D – F) melting curves measured by CD at 260 nm of 250 nM F-22G3 in the presence or absence of 40 μM NMM, 5 μM SST16 or 1 μM PhenDC3. NMM was incubated with F-22G3 during G4 …
Figure 7—figure supplement 3
Effects of ligands on F-22G3 FRET.
Representative FRET trajectories and heat maps of F-22G3 in the presence of NMM (A, B); 800 μM in folding reaction), SST16 (C, D); 5 μM) or PhenDC3 (E, F); 1 μM); n = 110 (B), n = 96 (D), n = 103 (F)…
Figure 7—figure supplement 4
Curve fitting parameters of the curves in Figure 7K.
Figure 7—figure supplement 5
Telomerase extension assays of F-22G3 in the presence of G4 ligands.
(A) Direct extension assays using 250 nM of F-22G3 or linear Bio-L-18GGG control, in the presence or absence of NMM (800 μM in folding reaction), SST16 (5 μM) or PhenDC3 (1 μM). For the reactions …
Figure 8 with 2 supplements
Telomerase extends ligand-stabilized tetrameric parallel G4.
(A), (B), (C) Telomerase extension assays using 1 µM of [7GGT]4 or a linear 7-mer control (or 250 nM in the reactions with PhenDC3), in the presence or absence of 40 µM NMM (A), 100 μM SST16 (B) or …
Figure 8—figure supplement 1
Demonstration of ligand-mediated stabilization of F-22G3.
(A – C) CD spectra and (D – F) melting curves measured by CD at 260 nm of 1 mM [7GGT]4 in the presence or absence of 40 μM NMM, 100 μM SST16 or 1 μM PhenDC3.
Figure 8—figure supplement 2
Telomerase extension of [7GGT]4 in the presence of titrations of G4 ligands.
Telomerase extension assays in the presence of either (A) NMM (0, 4 μM, 20 μM, 40 μM, 80 μM, 160 μM), (B) SST16 (0, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM) or (C) PhenDC3 (0, 0, 0.5 μM, 1 μM, 2.5 μM, 5 …
Figure 9 with 3 supplements
Telomerase unfolds ligand-stabilized tetrameric parallel G4.
(A), (D), (G) Representative single-molecule FRET trajectories of NMM-stabilized (A; 10 mM in folding reaction), SST16-stabilized (D; 100 µM) or PhenDC3-stabilized (G; 1 µM) F-[7GGT]4 in the …
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Figure 9—source data 1
Source data for the graph shown in Figure 9J.
- https://cdn.elifesciences.org/articles/56428/elife-56428-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
Effects of ligands on F-[7GGT]4 FRET.
Representative FRET trajectories (A, C, E) and heat maps (B, D, F) of F-[7GGT]4 in the presence of NMM (10 mM in folding reaction), SST16 (100 µM) or PhenDC3 (1 µM), without telomerase; n = 120 (B), …
Figure 9—figure supplement 2
Effect of ligands on FRET of F-[7GGT]4 in the presence of telomerase.
Representative FRET trajectories (A, C, E) and heat maps (B, D, F) of F-[7GGT]4 in the presence of NMM (10 mM in folding reaction), SST16 (100 µM) or PhenDC3 (1 µM), with telomerase; n = 80 (B), 60 …
Figure 9—figure supplement 3
Curve fitting parameters of the curves in Figure 9K.
Figure 10
Model for binding and extension of parallel G-quadruplexes by telomerase.
(A) Model of an extended conformation of the human telomerase reverse transcriptase (hTERT; grey) with a 10 nt RNA template (magenta) and the [7GGT]4 G-quadruplex (orange and dark red) docked into …
Author response image 1
Intramolecular (F-22G3) forming sequence (top) and hybridisation sequence (bottom).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293T (embryonic kidney, immortalized with adenovirus) | American Type Tissue Collection | Cat#: ATCC CRL-3216 | |
| Antibody | Anti-hTERT (sheep polyclonal) | Abbexa Ltd. | Cat#: abx120550 | IP (40 µg/ml) |
| Recombinant DNA reagent | pApex-CMV-hTERT (plasmid) | PMID:26158869 | Dr Tracy Bryan, Dr Scott Cohen | |
| Recombinant DNA reagent | pApex-CMV-dyskerin-U3-hTR (plasmid) | PMID:26158869 | Dr Tracy Bryan, Dr Scott Cohen | |
| Peptide, recombinant protein | hTERT amino acids 276–294 (peptide) | Abbexa Ltd. | Cat#: abx069990 | Sequence: ARPAEEATSLEGALSGTRH |
| Commercial assay or kit | Dynabeads M-280 Streptavidin | Thermo-Fisher | Cat#: 11206D | |
| Commercial assay or kit | NeutrAvidin Protein | Thermo-Fisher | Cat#: 31050 | |
| Commercial assay or kit | Unylinker CPG solid support | ChemGenes | Cat#: N-4000–05 | |
| Commercial assay or kit | Gel-Pak 2.5 Desalting Column | Glen Research | Cat#: 61-5025-25 | |
| Chemical compound, drug | AlexaFluor 555 NHS Ester | Thermo-Fisher | Cat#: A20009 | |
| Chemical compound, drug | (3-Aminopropyl)triethoxy silane | Alfa Aesar | Cat#: A10668 | |
| Chemical compound, drug | Biotin-PEG-SVA and mPEG-SVA | Laysan Bio | Cat#: BIO-PEG-SVA-5K-100MG and MPEG-SVA-5K-1g | |
| Chemical compound, drug | N-Methyl Mesoporphyrin IX (NMM) | Frontier Scientific | Cat#: NMM580 | |
| Chemical compound, drug | PhenDC3 | PMID:17260991 | Dr Marie-Paule Teulade-Fichou | |
| Chemical compound, drug | SST16 | PMID:19419877 | Dr Siritron Samosorn |
Additional files
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Supplementary file 1
Table of oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/56428/elife-56428-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56428/elife-56428-transrepform-v2.pdf
