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Published in final edited form as: Science. 2022 Feb 24;375(6585):1173–1176. doi: 10.1126/science.abn6840

Structural basis of human telomerase recruitment by TPP1-POT1

Zala Sekne 1,#, George E Ghanim 1,*,#, Anne-Marie M van Roon 1, Thi Hoang Duong Nguyen 1,*
PMCID: PMC7612489  EMSID: EMS142058  PMID: 35201900

Abstract

Telomerase maintains genome stability by extending the 3’ telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2 Å and 3.9 Å resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected DNA path exiting telomerase active site and a DNA anchor site on telomerase important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

Telomerase restores telomeric repeats ((TTAGGG)n in humans) by de novo DNA synthesis using a telomerase reverse transcriptase (TERT) subunit and an RNA template embedded within telomerase RNA (hTR in humans) (1). Telomerase activity is critical for the long-term proliferation of most cancers, germline and stem cells, while telomerase deficiency results in premature aging diseases (2). Human telomerase consists of two flexibly tethered functional lobes (3, 4): a catalytic core, in which TERT associates with the pseudoknot/template (PK/t) domain and conserved regions 4 and 5 (CR4/5) of hTR (5), and an H/ACA ribonucleoprotein (RNP) lobe, which is essential for telomerase biogenesis (6) (Fig. 1, A to C). The catalytic core also associates with a histone H2A-H2B dimer (4) (Fig. 1, A, D and E). Telomerase access to and activity at telomeres is tightly regulated (7). Mammalian telomeric DNA is bound and protected by a six-membered protein complex, called shelterin (8). The shelterin component TPP1 has been implicated in telomerase recruitment to telomeres (9–13). Within shelterin, TPP1 binds TIN2 and POT1, a single-stranded telomeric DNA binding protein (14, 15). TPP1-POT1 and TPP1-POT1-TIN2 complexes have been demonstrated to stimulate telomerase repeat addition processivity (RAP), the ability of telomerase to add multiple telomeric repeats with each DNA binding event (16, 17). Both RAP and interactions with shelterin are essential for telomerase function in vivo (9–11, 18). Yet, the structural basis of telomerase-shelterin interactions and shelterin-mediated telomerase processivity remains elusive.

Fig. 1. Structures of telomerase catalytic core with telomeric DNA and shelterin components.

Fig. 1

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(A) Domain organization of protein subunits in the catalytic core and TPT complex. Regions not observed in the structures are displayed as semi-transparent. Domain colors are used thorough unless indicated otherwise. (B) Secondary structure of hTR. (C) Schematic of human telomerase bound to TPP1 and POT1. (D) 3.2 Å cryo-EM reconstruction of telomerase catalytic core-DNA-TPP1 complex (fig. S6). (E) 3.9 Å cryo-EM reconstruction of telomerase catalytic core-DNA-TPP1-POT1 complex (fig. S7).

We prepared the human TPP1-POT1-TIN2 (TPT) complex (fig. S1A), demonstrated that it stimulated telomerase processivity in vitro (fig. S1, B and C), reconstituted its complex with human telomerase and the telomeric DNA substrate (T2AG3)5 (fig. S1, D and E), and verified the retention of the DNA within this complex (fig. S1F). Negative stain electron microscopy (EM) analyses showed that TPT binds telomerase catalytic core (fig. S2, A to D). Cryo-electron microscopy (cryo-EM) analyses showed conformational flexibility between the catalytic core, H/ACA lobe and TPT (figs. S2, E to G, S3, and S4, A to E). Focused classification/refinement resolved the DNA-bound telomerase catalytic core with either TPP1 or TPP1-POT1 at overall resolutions of 3.2 Å and 3.9 Å, respectively (Fig. 1, D and E; figs. S3, S5, S6, S7; Tables S1 and S2). TIN2 could not be resolved by image processing.

In both structures, the TPP1 OB-fold domain binds the bottom face of the telomerase catalytic core (Fig. 1, D and E). TERT has four domains: telomerase essential N-terminal (TEN) domain, telomerase RNA-binding domain (TRBD), reverse transcriptase (RT) domain and C-terminal extension (CTE) domain (Fig. 1A). TPP1 interposes between the TEN domain and a telomerase specific-insertion in fingers sub-domain (IFD) within the RT domain (Fig. 1D, right). In previous structures (3, 4), the TEN domain shows the most conformational variability. TPP1 binding induces conformational changes in the TEN domain and reduces its flexibility, thereby improving its local resolution (figs. S5G and S8A). We also observe a slight compaction of the CTE domain and the P2 stem of hTR (fig. S8, B and D).

The 3.2 Å telomerase-DNA-TPP1 structure reveals the molecular basis of TPP1-telomerase interactions (Fig. 2; and fig. S6, I to K). Previous studies identified an N-terminal region of TPP1 OB-fold domain (NOB) and a TPP1 glutamate and leucine-rich (TEL) patch that are crucial for stimulating telomerase processivity in vitro and telomerase recruitment to telomeres in vivo (fig. S9B) (9–11, 13). The NOB and TEL-patch form three areas of contact with TERT (Fig. 2A). The NOB seats into a hydrophobic cleft formed by the TEN domain and the IFD, in contrast to its extended conformation in the crystal structure of the isolated TPP1 OB-fold domain (16) (Fig. 2, B and C, and fig. S6I). The glutamate-rich region of the TEL-patch (residues 168-172) folds into a short α-helix that is accommodated by a basic surface on the TEN domain (Fig. 2D and fig. S6J). The other region of the TEL-patch (residues 210-215, R180 and L183) interacts extensively with the TEN domain and IFD (Fig. 2E and fig. S6K). Superimposition of the unbound versus telomerase-bound TPP1 OB-fold structures shows that the TEL-patch loop (residues 212-215) has some flexibility to enable interaction with TERT (Fig. 2B). Mutations in the NOB or the TEL-patch severely disrupt TPP1-telomerase interactions and recruitment to telomeres (9–11). Thus, the interactions observed here are functionally important for telomerase recruitment by TPP1.

Fig. 2. Telomerase-TPP1 interactions.

Fig. 2

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(A) Interactions between TPP1 OB-fold domain and TERT. Circles indicate the three contacts that the NOB (orange) and TEL-patch (red) of TPP1 make with TERT. The β-hairpin, which become ordered upon TPP1 binding, is highlighted. (B) Comparison of the unbound (grey, PDB 2I46 (16)) and telomerase-bound (colored) OB-fold domain of TPP1. (C) Close-up view of the interactions between the NOB of TPP1 and TERT (fig. S6I). (D and E) Close-up views of the interactions between the TEL-patch and TERT (fig. S6, J and K).

Our structure reveals the key TERT regions necessary for TPP1 interaction. Helix α5 (residues 122-135) extensively interacts with TPP1 NOB and TEL-patch (Fig. 2, C to E; and fig. S6, I to K). Upon binding to TPP1, the β3-β4 hairpin and loop 60-76 of TERT become ordered (Fig. 2A and fig. S8A). Both helix α5 and the ß3-ß4 hairpin are located in the N-terminal dissociates-of-activities domain of telomerase (N-DAT) embedded in the TEN domain (19). Mutations in the N-DAT were shown to have small effects on telomerase catalytic activity but impaired telomere elongation in vivo, suggesting defects in telomerase recruitment to telomeres (19, 20). Additional contacts were observed between TEN domain K78, helix α3 (residues 44-46) and the TPP1 TEL-patch (Fig. 2, D and E; and fig. S6J). Our findings explain previous studies which showed that charge-reversal mutations at R132 and K78 of the TEN domain significantly reduced the RAP stimulation by TPP1-POT1 and disrupted telomerase localization to telomeres in vivo. These defects were rescued by a charge-swap E215K mutation in TPP1 (12, 21). The IFD has also been suggested to be involved in TPP1 binding and telomerase recruitment to telomeres (22). In our structure, the base of an extended β-sheet formed by the IFD (residues 766, 767, 797-799) and the TEN domain hold the NOB in place (Fig. 2C, and fig. S6I). Additionally, part of the IFD helix α25 (residues 771-775) is sandwiched between the two contact sites in the TEL-patch (Fig. 2D).

POT1 is more flexibly engaged with telomerase than TPP1, evident from its local resolution range of 7-9 Å (figs. S4C, S4D, and S5H). The 3.9 Å telomerase-DNA-TPP1-POT1 map allowed us to rigid-body fit a DNA-bound POT1 crystal structure (POT1 OB1 and OB2) (Fig. 1, A and E; Fig. 3; and figs. S7F, and S9C) (23). OB3 and the Holliday-junction resolvase-like (HJRL) domains of POT1 and the C-terminal region of TPP1, which interacts with TIN2 and POT1, are unresolved in our structures (Fig. 1A) (24, 25). With high-affinity for single-stranded DNA (23), POT1 was thought to associate with telomerase via TPP1 and enhance telomerase RAP by increasing avidity to telomeric DNA (16, 26, 27). Surprisingly, POT1 binds not only the DNA substrate but also TERT and forms a gate in front of telomerase active site (Fig. 3). The N-DAT within the TEN domain wedges between the two OB-fold domains of POT1 (Fig. 3B). Substitutions of POT1-contacting residues in the N-DAT severely affect telomere elongation in vivo with minimal effects on telomerase catalytic activity in vitro (19). Thus, the identified interactions between POT1 and telomerase in our structure are crucial for telomerase recruitment to telomeres in vivo.

Fig. 3. Telomerase-POT1 interactions.

Fig. 3

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(A) Structure of telomerase-DNA-TPP1-POT1 complex. (B) 3.9 Å cryo-EM reconstruction of telomerase-DNA-TPP1-POT1 complex. The inset highlights interactions between POT1 and TERT. The N-DAT region (purple) interacts with both TPP1 and POT1.

We previously used a T12(T2AG3) DNA substrate and observed only the 3’ terminal TTAGGG repeat (4). Here, we used a longer (T2AG3)5 DNA substrate to allow for TPP1-POT1 binding. Unlike its ciliate TEBPβ homolog (28), TPP1 does not contact the DNA (fig. S10). Yet, its association with telomerase stabilizes the DNA, allowing us to resolve another TTAGGG repeat in our 3.2 Å telomerase-DNA-TPP1 map (fig. S6G). The telomerase-DNA-TPP1-POT1 map shows additional DNA density, forming a continuous path between the 12-nucleotide (12-nt) modelled DNA and the DNA bound to POT1 from the previous crystal structure (PDB 1XJV) (23) (fig. S7K). Although the resolution of this region is insufficient for de novo modelling, we use this density and the two DNA models to propose the DNA path through telomerase. The DNA 5’ end threads through a positively charged tunnel formed by POT1 OB1 domain and the TEN domain, and is guided along the TEN domain by POT1 OB2 domain (Fig. 4, A and H; and fig. S11, A and B). The DNA then makes a sharp turn and traverses the IFD and CTE domains before reaching telomerase active site (Fig. 4A; and figs. S6G, S7G).

Fig. 4. Telomeric DNA substrate.

Fig. 4

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(A) The DNA path guided by TERT, hTR and POT1. The left upper panel shows how the view is related to the telomerase-DNA-TPP1-POT1 reconstruction. (B) Interactions between the template RNA and the DNA upstream of the RNA template-DNA duplex (fig. S6L). (C) Close-up view of the interactions between the DNA substrate and the proposed anchor site on the TEN domain and the IFD (fig. S6N). (D) Interactions between the flipped-out G23 DNA base and TERT (fig. S6M). (E) Telomerase activity assays in the absence and presence of the TPT complex for wild-type (WT) enzyme and TERT mutants at the proposed DNA anchor site (Y176A/Q177A, K757A/F759A and Q794A) (fig. S6, H and N). Assays were performed after enzyme enrichment on MagStrepXT resins using ZZ-TEV-twin-Strep tagged TERT. RC, recovery control. Experiments were performed in triplicate. Detailed analyses of the input lysates are shown in fig. S12. (F and G) Bar graphs showing the quantifications of telomerase activity and processivity of the activity assays shown in (E), respectively. Values were normalized to telomerase without TPT. Error bars are the standard errors of the mean (SEM) obtained from the replicates. (H) Schematic of the DNA substrate and its interactions with TERT, hTR and POT1. Only interactions with the 5’ part of the DNA following the DNA-RNA duplex are highlighted as interactions of the DNA-RNA duplex with TERT have been discussed previously (4).

The telomeric DNA path reveals a previously undiscovered DNA threading surface on the TEN domain and the IFD of TERT, with implications for telomerase processivity. The TEN domain is essential for RAP and telomerase recruitment to telomeres (12, 29, 30). Several studies have suggested that the TEN domain contains an anchor site that binds the single-stranded DNA substrate and prevents DNA dissociation, thereby promoting RAP (20, 30–34). However, the molecular determinants for the DNA binding activity of the TEN domain and the location of the anchor site remained unclear. We identified a PLYQ motif in the TEN domain (residues 174-177), which binds and turns the DNA substrate towards the TEN-POT1 interface (Fig. 4C, and figs. S6N, S7I, S11A and S11B). This motif is highly conserved in vertebrates (fig. S9A). Binding of the TEN domain to the DNA is assisted by the IFD (Q794 and β17, residues 752-759) (Fig. 4C).

To test the role of the PLYQ motif and the IFD as contributors to the DNA anchor site, we reconstituted telomerase with TERT Y176A/Q177A, K757A/F759A and Q794A mutations, which significantly reduced telomerase activity and RAP (Fig. 4, E to G; and fig. S12). Addition of the TPT complex rescued the RAP defects in these mutants (Fig. 4, E to G). Similar observations were made previously with Y176A and Q177A single mutations (10, 26, 30). Substitutions at TERT residues 170-175 significantly increase the K m for (T2AG3)3 primer and affect telomere elongation in vivo (19, 30). This suggests that the RAP defect observed in the mutants is caused by DNA binding defects during repeat synthesis, and the TPT complex rescued the RAP defects by compensating for the reduced DNA affinity. Thus, we propose that the PLYQ motif of the TEN domain and strand β17 of the IFD form the anchor site that is crucial for DNA retention during RAP.

hTR also contributes to shaping the DNA path. Immediately downstream of the four base-pair DNA-RNA template duplex, the RNA template base A54 stabilizes the flipped-out DNA base T26 by base-stacking, and coordinates the phosphate backbone of the neighboring T25 together with the RNA template base A55 (Fig. 4, A, B and H; and fig. S6L). Previous studies showed that the RNA template mutants A54U and A55U compromised telomerase RAP (18), suggesting that these unexpected DNA-RNA template interactions may be important for - RAP. We also observe another flipped-out DNA base (G23), which is stabilized by the IFD and CTE domain (Fig. 4, D and H; and fig. S6M).

Tetrahymena p50-TEB complex is functionally comparable to human TPP1-POT1. While TPP1-POT1 is part of the shelterin complex residing at mammalian telomeres, p50-TEB complex is integral Tetrahymena telomerase holoenzyme subunits (35, 36). In Tetrahymena telomerase (36), p50 occupies a similar position to TPP1 relative to the TEN domain (fig. S11E). However, POT1 arrangement is strikingly different from that of TEB (fig. S11E). The DNA substrate in Tetrahymena does not travel along the TEN domain as observed in our structure (fig. S11E). The positive charge of the DNA threading surface on the human TEN domain is not conserved in Tetrahymena and S. cerevisiae (fig. S11, C and D). Therefore, the DNA path along the human TEN domain may be vertebrate-specific.

Finally, our structures explain how TPP1 and POT1 facilitate telomerase association with telomeric DNA and stimulate telomerase RAP. With each round of telomeric repeat synthesis, the product DNA can either realign with the template for another round of repeat synthesis or dissociate. In telomerase structure alone (4), the TEN domain is conformationally flexible, resulting in unstable DNA binding, increased DNA dissociation during repeat synthesis and thus lower RAP (fig. S13A). TPP1 binding stabilizes the TEN domain and allows the anchor site to engage the DNA substrate more stably. POT1 further stabilizes DNA binding by promoting the DNA-TEN domain interaction. Together, TPP1 and POT1 cooperatively increase RAP by reducing DNA dissociation during repeat synthesis, agreeing with previous kinetic studies (37) (fig. S13B). The observed flexibility of POT1 may be necessary for efficient translocation during RAP and/or for accommodating G-quadruplex structures formed by the telomeric DNA product (27) (fig. S4F). Recruitment by shelterin (via TPP1-POT1) would ensure high processivity of telomere-engaged telomerase. Telomerase recruited to a telomere without shelterin would dissociate more easily, thus impairing processive DNA synthesis.

Supplementary Material

Supplimentary Materials

One-Sentence Summary.

Structures of human telomerase with recruitment factors reveal the molecular basis of telomerase recruitment to telomeres.

Acknowledgments

We thank the MRC-LMB EM facility staff for access and support of electron microscopy sample preparation and data collection; J. Grimmett and T. Darling for maintaining the computing facility; C. Johnson for assisting with biophysical characterization; A. Carter, K. Collins, R. Hegde, D. Rio and S. Scheres for critical reading of the manuscript; the Nagai, Löwe, Passmore and Scheres labs for sharing reagents, equipment and technical advice; B. Greber for sharing a script to calculate conformational changes; D. Barford, K. Collins, E. Nogales and S. Scheres for their support; T. Croll for advice with ISOLDE, E. Zhong for advice with CryoDRGN.

Funding

THDN is supported by a UKRI-Medical Research Council grant (MC_UP_1201/19).

GEG is supported by a Jane Coffin Childs Postdoctoral Fellowship.

Footnotes

Author contributions:

T.H.D.N. initiated and supervised the project. Z.S. and G.E.G. purified samples, prepared EM grids, collected and analyzed EM data, performed model fitting and all biochemical experiments and quantification. G.EG. performed model building and refinement. A.M.v.R. prepared telomerase extracts and performed model refinement. Z.S, G.E.G. and T.H.D.N. analyzed the structures. Z.S., G.E.G., A.M.v.R and T.H.D.N. wrote the paper.

Competing interests: Authors declare that they have no competing interests.

Data and materials availability

Cryo-EM maps of telomerase-DNA-TPP1 are deposited with the Electron Microscopy Database under accession numbers EMD-14196 (sharpened) and EMD-14198 (unsharpened). Cryo-EM maps of telomerase-DNA-TPP1-POT1 are deposited with the Electron Microscopy Database under accession numbers EMD-14197 (sharpened) and EMD-14199 (unsharpened). PDB coordinates are deposited with the Protein Data Bank under accession number PDB ID 7QXA for telomerase-DNA-TPP1 complex, 7QXB (Cα-POT1) and 7QXS (POT1 with side-chains) for telomerase-DNA-TPP1-POT1 complex. Materials are available from T.H.D.N. under a material transfer agreement with the MRC-Laboratory of Molecular Biology. Correspondence should be addressed to T.H.D.N (knguyen@mrc-lmb.cam.ac.uk) or G.E.G (gghanim@mrc-lmb.cam.ac.uk).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplimentary Materials
EMS142058-supplement-Supplimentary_Materials.docx (40.6MB, docx)

Data Availability Statement

Cryo-EM maps of telomerase-DNA-TPP1 are deposited with the Electron Microscopy Database under accession numbers EMD-14196 (sharpened) and EMD-14198 (unsharpened). Cryo-EM maps of telomerase-DNA-TPP1-POT1 are deposited with the Electron Microscopy Database under accession numbers EMD-14197 (sharpened) and EMD-14199 (unsharpened). PDB coordinates are deposited with the Protein Data Bank under accession number PDB ID 7QXA for telomerase-DNA-TPP1 complex, 7QXB (Cα-POT1) and 7QXS (POT1 with side-chains) for telomerase-DNA-TPP1-POT1 complex. Materials are available from T.H.D.N. under a material transfer agreement with the MRC-Laboratory of Molecular Biology. Correspondence should be addressed to T.H.D.N (knguyen@mrc-lmb.cam.ac.uk) or G.E.G (gghanim@mrc-lmb.cam.ac.uk).

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