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Structure of telomerase-bound CST with Polymerase α-Primase

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3’-overhang of the G-strand and a group of proteins called shelterin1,2. Among these, TPP1 and POT1 associate with the 3’-overhang, with POT1 binding the G-strand3 and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT)4. The ends of the telomeric DNA are replicated and maintained by telomerase5, for the G-strand, and subsequently DNA Polymerase α-Primase6,7 (PolαPrim), for the C-strand8. PolαPrim is stimulated by CTC1–STN1–TEN1 (CST)9–12, but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex—telomerase core RNP, p50/TPP1, CST and PolαPrim—that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.

Synthesis of the G-strand at the ends of telomeres by telomerase is terminated by the heterotrimeric complex CST 12,13 . CST is essential for maintaining the telomere C-strand and also plays a role in overcoming genome-wide replication stress 9,14,15 . Like the shelterin proteins and telomerase, mutations in CST lead to telomere biology disorders 16 including Coats Plus and dyskeratosis congenita 17 . CST small subunits STN1 and TEN1 are closely related to those in replication protein A (RPA) 18 , a singlestranded DNA binding protein involved in all aspects of DNA replication and repair, while the large subunit Ctc1/CTC1/Cdc13 is more diverse [19][20][21][22][23][24] . Structural and biochemical studies of CST proteins have suggested various stoichiometries, oligomerization states, and functions of subunits 19,20, 24 . The only structure of complete CST 19 , from human, revealed a decameric architecture of heterotrimers in the presence of single-stranded telomeric (G-strand) DNA (sstDNA). CST is proposed to inhibit telomerase activity by physical interaction with shelterin proteins TPP1-POT1 at telomere ends 12,25,26 and G-strand sequestration 12,13 , and it promotes C-strand fill-in by association with PolαPrim 27-30 , but structures of these interactions are lacking. PolαPrim is an unusual polymerase containing both primase and DNA polymerase subunits; the primase synthesizes an RNA primer on a DNA template and then hands off the duplex to the polymerase that initiates synthesis of a short DNA duplex 6,7 .
Tetrahymena telomerase holoenzyme comprises, in addition to its RNP catalytic core of TERT, telomerase RNA (TER) that provides the template for G-strand telomere repeat synthesis, and LARP7 assembly protein p65, several proteins that are orthologous to human proteins that only transiently associate with telomerase at telomeres 31,32 . These include p50, the structural and functional equivalent of TPP1 that recruits and activates telomerase 23,33,34 ; Teb1, a subunit of a trimeric RPA related complex TEB 23 , that binds the sstDNA 35 and together with p50 increases activity and processivity like its ortholog POT1 [36][37][38] ; and another trimeric RPA related complex p75-p45-p19 that has been identified as Tetrahymena Ctc1-Stn1-Ten1 (TtCST) 23,24 . The constitutive association of these proteins with telomerase catalytic core makes Tetrahymena telomerase an ideal model system for elucidating details of the protein structures and interactions that regulate G-strand and C-strand synthesis 23,39,40 . Structural studies of Tetrahymena telomerase and PolαPrim described here show how monomeric TtCST binds p50/TPP1 and PolαPrim on different interfaces to coordinate G-strand termination and C-strand fill-in synthesis, and suggest commonalities with interactions at human telomeres.

Overall structure of telomerase bound CST
While cryo-EM studies of Tetrahymena telomerase have provided high-resolution structures of the RNP catalytic core, TEB heterotrimer, and p50 OB 40 , the dynamic positioning of TtCST has limited its structure modeling to date. Here, we combined three previously reported datasets of Tetrahymena telomerase bound to sstDNA 40 and conducted focused classification on TtCST followed by refinement of the holoenzyme to obtain a reconstruction with an overall resolution of 3.5 Å (Fig. 1a, Extended Data  Table 1). For the model of TtCST, Stn1-Ten1 crystal structures 23,24 were rigid body fit into the density and manually refined with little change, and Ctc1 was built de novo (Fig. 1b, Extended Data Fig. 2). Modeling of the N-terminal domain of Ctc1, which has lower resolution (Extended Data Fig. 1c), was facilitated by using information derived from NMR data on secondary structure elements and inter-β-strand NOEs (see Methods) (Extended Data Fig. 3). Ctc1, whose domain structure was not previously established, comprises three OBs (OB-A, -B, and -C) connected by structured linkers that stabilize the rigid pairwise interactions between the domains (Fig. 1c, d). Ctc1 OB-C has a C-shaped cleft and Zn-ribbon motif (Fig. 1c) typical of the C-terminal OB of the large subunit of RPA 18 and related complexes, including mammalian CTC1 19 , POT1 37,38 , and TtTeb1 35,40 .
The tandem winged helix-turn-helix (WH-WH) domain of Stn1 is connected to OB by a flexible linker and is not visible in the cryo-EM map, consistent with its multi-positioning shown by negative-stain EM 23 . Overall, our structure of monomeric TtCST strongly suggests its origin from RPA and establishes the domain structure of the least conserved subunit Ctc1.

Flexible interface between Ctc1 and p50
p50 has an N-terminal OB and a C-terminal domain that is invisible in the cryo-EM map (Fig. 1a). p50/TPP1 OB interacts with telomerase on TERT TEN and TRAP domains 39-42 , but how TPP1-POT1 interacts with CST is unknown 25,26 . We find that TtCST is anchored on p50 via Ctc1 OB-A (Fig. 1c). In the structure, TtCST is positioned across the top of the TERT ring (Fig. 1b), and stabilized in this predominant conformation by additional interactions between Ctc1 and TERT-TER catalytic core (Extended Data Fig. 2h-k). However, these are not stable interactions, as other conformations resolved by 3D classification show TtCST hinged away from TERT (Fig. 2a, Extended Data Fig. 1a). In the cryo-EM map, a previously uncharacterized density of p50 protrudes from its OB C-terminus into Ctc1 OB-A (Extended Data Fig. 1g). p50 residues 185-208 were built against the density as helix α5 and strand β7, the latter of which forms an extended β sheet with β1-β4-β5' of Ctc1 OB-A (Fig. 2b). However, previous biochemical studies showed that p50 C-terminal truncation at residue 213 almost abrogates binding with Ctc1, while truncation at residue 252 showed binding with Ctc1 comparable to full-length protein 43 .
We therefore investigated whether p50 residues between 208-255 might be contributing to the binding interface with Ctc1. We made a series of p50 peptides and monitored their interaction with Ctc1 OB-A by NMR (Extended Data Fig. 4). 1 H-15 N HSQC spectra show that optimal binding requires residues 228-250 (Fig. 2c, Extended Data Fig. 4b). This peptide forms a 1:1 complex with Ctc1 OB-A that is in slow exchange on the NMR timescale indicating slow off-rate (Extended Data Fig. 4c). Talos+ (ref 44 ) secondary structure scores, CS-Rosetta 45 modeling, and chemical shift mapping indicate that p50 peptide residues 228-241 form a β-hairpin that interacts with the Ctc1 OB-A β-barrel near β1-β2 linker, β4, and β5 (Fig. 2d, Extended Data Fig. 4e-g). Together, these cryo-EM and NMR data define a Ctc1 binding motif (CBM) adjacent to p50 OB that tightly associates with Ctc1 but allows hinging movement of the entire CST complex on p50.

Overall structure of PolαPrim with CST
A defining feature of CST is its ability to recruit PolαPrim for C-strand synthesis; however, the interface between PolαPrim and CST has not been structurally characterized in any organism 9,27-30, 46 . To both verify that p75-p45-p19 is functionally TtCST and define the mechanism of PolαPrim recruitment, we assembled Tetrahymena telomerase-PolαPrim complex using endogenously expressed telomerase and recombinant PolαPrim in the presence of sstDNA d(GTTGGG)10, and determined its cryo-EM structure ( Fig. 3a-c, Extended Data Fig. 5, 6). In the telomerase holoenzyme PolαPrim binds TtCST in the absence or presence of sstDNA (Fig. 3c). Since the entire TtCST-PolαPrim complex was flexibly positioned relative to p50, as seen for TtCST alone, the TtCST-PolαPrim and telomerase RNP core-TEB-p50 complexes were processed separately to obtain 3D reconstructions of 4.2 Å and 2.9 Å resolution, respectively (Fig. 3a, Extended Data Fig. 6, Extended Data Table 1). The modeled structures are the first of CST-bound PolαPrim and the highest resolution structure of telomerase RNP core to date (Fig. 3b, Extended Data Fig. 6f). Modeling of TtCST in the complex by initial rigid body fitting of the structure determined in the absence of PolαPrim revealed the presence of additional density on Ctc1 OB-B and OB-C that could be fit with the crystal structure of Stn1 WH-WH 23,24 (Fig. 3b, Extended Data Fig. 6f). Binding of PolαPrim to TtCST displaces Ctc1 from its stable position across the top of the TERT ring (compare Fig. 3a, b to Fig. 1a, b), and instead POLA1 is positioned near TER loop 2. The Stn1 WH-WH binding site on Ctc1 would also be occluded in the stable conformation of TtCST on p50-TERT in the absence of PolαPrim.
However, only the catalytic POLA1core was well resolved in the cryo-EM map (Fig. 3a, d). The rest of PolαPrim appears as a fuzzy density connected to POLA1core opposite the interface with Ctc1 (Extended Data Fig. 5f). Therefore, we also investigated the cryo-EM structure of PolαPrim alone, and  Fig. 8c) with the active site on POLA1core sterically blocked by POLA1CTD and POLA2 for DNA entry 48,49 . Here, our studies provide the first structures of a PolαPrim compatible with activity, and they establish its direct interactions with TtCST, as described in detail next.

CST interaction with POLA1 and sstDNA
POLA1core comprises an N-terminal domain (NTD) that brackets a catalytically dead exonuclease (Exo), and a C-terminal DNA polymerase that contains palm, fingers, and thumb domains 7 (Fig. 4a, b). All elements of POLA1core except the tip of the thumb are well defined in the cryo-EM map. POLA1core interacts with CST via its Exo and C-terminal region of NTD, which positions its active site ~40 Å from the interface (Fig. 4a). CST interacts with POLA1core via Ctc1 OB-C and Stn1 (Fig. 4b-d). On Ctc1 OB-C, the conserved Zn-ribbon motif interacts primarily with conserved Exo β11-β12 hairpin (Fig. 4c, Extended Data Fig. 8d). Ctc1 helix α14 and Stn1 OB β1-β2 and β3-β4 loops form a binding pocket that accommodates POLA1core NTD helix α19 (residues 731-748) with charge complementarity to its side chains (Fig. 4c, Extended Data Fig. 8e-g). Helix α19 is a flexible loop in all other structures of PolαPrim [48][49][50][51]53 , and apparently becomes structured only on binding Ctc1-Stn1 (Extended Data Fig.   8e), indicating the importance of this interaction for CST binding. Structure-based sequence alignment suggests conservation of these interfaces on POLA1core across a wide-range of species (Extended Data   Fig. 8g). Behind this interface, the three helices of Stn1 WH2 are inserted into a gap between Ctc1 OB-B and POLA1 Exo (Fig. 4d).
Cryo-EM density for ~10 nucleotides of sstDNA is observed on Ctc1 OB-C across the C-shaped binding cleft near the Zn-ribbon (Fig. 4a, c). Substitution of three conserved residues R395E, Y445A and F473A on the binding surface almost abolishes sstDNA binding to purified TtCST, as determined by electromobility shift assays (EMSA) with d(GTTGGG)5 (Fig. 4e, f). The sstDNA extends 5'!3' into an entry port, formed by POLA1core NTD and Exo and Ctc1 OB-C (Extended Data Fig. 8h), of a highly basic channel that leads to the active site of POLA1core, where the primer-sstDNA duplex would bind The structure shows that POLA1core Exo and NTD form an extensive interface with TtCST involving Ctc1 OB-C, Stn1 OB, and Stn1 WH1, which is otherwise flexibly tethered to Stn1 OB in the absence of PolαPrim. The sstDNA on Ctc1 OB-C appears positioned for entry into a template (G-strand) binding tunnel on POLA1core, and there is weak density within the tunnel that we attribute to sstDNA (Extended Data Fig. 8i, j), suggesting the possibility that this structure has captured the polymerase mode after handoff from primase. Although no DNA-RNA or DNA-DNA duplex is present, there is unassigned density between POLA1core palm and thumb (Fig. 3a) that fits the dimensions of a G-quadruplex formed by four Tetrahymena telomere repeats (Extended Data Fig. 8j). We propose that the G-quadruplex may have serendipitously trapped the PolαPrim in an incipient DNA polymerization state.

Comparison to human CST
While Tetrahymena Stn1 and Ten1 have the same domain structure as human STN1 and TEN1, respectively, human CTC1 (hCTC1) is much larger than Tetrahymena Ctc1, with seven OBs (OB-A through OB-G) 19 (Fig. 5a

Coordinated synthesis of G-and C-strands
Co-ordinated synthesis of telomeric G-and C-strands is orchestrated by interactions between telomerase, shelterin proteins TPP1 and POT1, sstDNA, CST, and PolαPrim, whose molecular details have been largely undefined. Taking advantage of the constitutive association of p50/TPP1, Teb1/POT1, and TtCST with Tetrahymena telomerase along with previous structural studies of telomerase we determined structures and interfaces between all of these components. TtCST Ctc1 Cterminal domain OB-C and Stn1 bind to PolαPrim POLA1 and Ctc1 N-terminal domain OB-A binds p50/TPP1 (Fig. 5d). p50/TPP1 OB in turn binds to TERT TEN-TRAP domains, constitutively in Tetrahymena but transiently to recruit telomerase to telomeres in humans 2, 42 . Biochemical studies have shown that CST binds G-strands released from telomerase during telomere repeat synthesis 12,13 . Our structures show that TtCST binds p50 on a flexible hinge (CBM), placing it in proximity to where the 3'-end of sstDNA released from the TER template would be. Teb1, that also interacts with p50, could contribute by initially maintaining a hold on the 5' exiting DNA, until it binds TtCST. C-strand synthesis requires that the released 3'-end of the G-strand enters the active site of the primase, where a short RNA primer is synthesized. PRIM2c binds the RNA-DNA duplex and hands it to the active site of POLA1 (Fig. 5d). Our structure explains how CST enhances the activity of POLA1, by binding the Gstrand and feeding it into the entry port for the POLA1 template channel (Fig. 4g), thereby unwinding and preventing the reformation of any G-quadruplexes 55       To determine TtCST structure, telomerase particles selected from three published datasets (the T3D2, T4D4 and T5D5 datasets as detailed in ref 40 ) were combined, resulting in over 2.5 million good particles (Extended Data Fig. 1a). Refinement of these particles without a mask generated a reconstruction with only weak density for TtCST, which confirms the multiple orientation of TtCST relative to the rest of telomerase holoenzyme 23 . To separate particles with TtCST at different positions, an alignment-free 3D classification was performed using a spherical mask covering the TtCST region (mask1). Particles from classes with TtCST at similar positions were grouped together and refined, resulting in three reconstructions (P1, P2 and P3 in Extended Data Fig. 1a), among which P1 has the best density and the largest number of particles. To improve the overall density of P1, we performed another round of 3D classification with local angular search (RELION options: --sigma_ang 8 --healpix_order 4). A soft mask (mask2) was used in this step to exclude the flexible p65. 259,330 particles from the best class were selected for 3D refinement, following by refinement of contrast transfer function (CTF) parameters and Bayesian polishing in RELION. The resulting "shiny" particles were refined to 3.5 Å resolution for the entire telomerase holoenzyme including TtCST (Extended Data Fig. 1b-d). An additional focused 3D classification step was conducted to improve the local resolution of Ctc1 OB-A. The resulting 78,471 particles were refined to 3.8 Å resolution using mask2.
For the newly collected telomerase-PolαPrim and PolαPrim datasets, dose-fractionated frames of each movie were 2x binned (pixel size of 1.1 Å), aligned for the correction of beaminduced drift, and dose weighted using RELION's implementation of UCSF MotionCor2 (ref 63 ).
CTF parameters, including defocus and astigmatism, of each dose-weighted micrograph were determined by CTFFIND4 (ref 64 ) within RELION.
Two datasets of telomerase-PolαPrim, one for each data collection session, were initially processed separately (Extended Data Fig. 6a). Particles picked from 2,000 representative micrographs using template-free auto-picking in RELION were screened by 2D classification, and the best particles were selected to train a particle detection model in Topaz 65 for subsequent neural-network based particle picking for all micrographs. After several rounds of 2D and 3D classifications as detailed in Extended Data Fig. 6a, good particles selected from two datasets were combined, resulting in over 1.6 million particles. Refinement of these particles without using any mask generated a reconstruction with only weak density for TtCST-PolαPrim, suggesting that TtCST-PolαPrim also has multiple orientations relative to the rest of telomerase holoenzyme, including telomerase core RNP, TEB heterotrimer and p50, as previously observed for the TtCST dataset (Extended Data Fig. 1a). Therefore, these two parts were processed separately in the following steps. For telomerase core RNP-TEB-p50, a soft mask was used to exclude the dynamic TtCST-PolαPrim during 3D refinement, which resulted in a 3.1 Å resolution reconstruction. After an additional round of focused classification with local angular search in 8° (RELION options: --sigma_ang 8 --healpix_order 4), 539,078 particles from the best class were selected and refined to 2.9 Å resolution (Extended Data Fig.   6b, c). For TtCST-PolαPrim, three rounds of alignment-free 3D classification with an optimized regularization parameter (RELION option: --tau2_fudge 16) were performed in parallel using a spherical mask covering the TtCST-PolαPrim region (mask2). 427,158 particles from classes with interpretable TtCST-PolαPrim densities were combined after removing duplicates.
Refinement of these particles generated a reconstruction with clear TtCST-PolαPrim density.
Then, we shifted the center of each particle to TtCST-PolαPrim and performed signal subtraction using mask4 to only keep the signal from TtCST-PolαPrim. After two rounds of 3D classification using mask4, 142,912 particles were selected and refined using the same mask, which resulted in a 4.2 Å resolution reconstruction for TtCST-PolαPrim (Extended Data Fig.   6b, d). These particles were back projected to original particles without signal-subtraction and refined to 4.4 Å resolution for the entire complex including both telomerase and TtCST-PolαPrim.
For the PolαPrim dataset (Extended Data Fig. 7), particle picking was conducted using Topaz 65 in a similar way as described above for the PolαPrim bound telomerase datasets.
After two rounds of 2D classification, 1.3 million particles were selected and refined using an initial model generated by cryoSPARC 66 . The resulting cryo-EM map has a "head" that is the catalytic core domain of POLA1 and a bow-tie-shaped "body" that contains POLA2, PRIM1, PRIM2N and the C-terminal domain of POLA1. The "head" and the "body" have multiple orientations relative to each other as indicated by their low-resolution densities in the 3D reconstruction and the 2D classification results (Extended Data Fig. 7b). Focused refinement of the "head" didn't work well due to its small size, but focused refinement of the "body" generated a map with clear secondary structure features. After one round of 3D classification using the same mask for the "body", 264,498 particles were selected and refined to 4.5 Å resolution.
During the 3D classification step, a notable hinge movement was observed within the "body".
Due to that, we further refined the two halves of the "body" individually and obtained a 4.0 Å resolution reconstruction for POLA2-POLA1CTD-PRIM2N and a 4.3 Å resolution reconstruction for PRIM2N-PRIM1 (Extended Data Fig. 7c-e).
All cryo-EM maps were sharpened with a negative B-factor and low-pass filtered to the stated resolution using the relion_postprocess program in RELION. Local resolution evaluations were determined by ResMap 67 with two independently refined half-maps. Data collection and processing statistics are given in Extended Data Table 1.

Model building and refinement. For the modeling of TtCST, crystal structures of
Tetrahymena Ten1-Stn1-OB 23,24 (PDB 5DOI and 5DFM) and cryo-EM structure of Tetrahymena telomerase RNP-TEB-p50 40 (PDB 7LMA) were initially rigid-body fitted into the 3.5 Å resolution cryo-EM map (Extended Data Fig. 1c) using UCSF Chimera 68 , and refined manually in COOT 69 . Density of Ctc1 was traced from its signature C-terminal α helix, and models of OB-C and OB-B were built de novo against the density in COOT. Visible densities of amino acid residues with bulky side chains, such as Phe, Tyr and Trp were used as guidance for sequence assignment (Extended Data Fig. 1f). For Ctc1 OB-A, an initial model was built against the cryo-EM density and refined manually with structure information obtained from NMR (Extended Data Fig. 3). Briefly, secondary structure information obtained from TALOS+ (ref 44 ) was used to define the boundaries of β strands within the OB (Extended Data Fig. 3d), and 105 inter-β-strand NOE restraints were used to refine the relative position of the β strands (Extended Data Fig. 3e). Lastly, p50 residues 184-208 following the C-terminus of the previous p50 OB model (PDB 7LMA) were built into the cryo-EM map adjacent to Ctc1 OB-A (Extended Data Fig. 1g). All models were refined using Phenix 70 in real space with secondary structure, Ramachandran, and rotamer restraints. Refinement statistics of the models are summarized in Electrophoretic mobility shift assay (EMSA). Tetrahymena CST-p50 and its mutants were expressed and purified from insect cells as described above. Radioactive 32 P 5'-labeled (GTTGGG)10 DNA primer was used for EMSAs. TtCST-p50 proteins were incubated with DNA primer in 10 µl EMSA buffer [20 mM HEPES-NaOH (pH 8.0), 150 mM NaCl, 2 mM MgCl2, 5% glycerol, and 1 mM TCEP] for 1 hour on ice before being loaded onto a 0.5× TBE 0.7% agarose gel. The gel was vacuum dried and exposed to a phosphor imaging screen for overnight. The final image was obtained by scanning the screen on a ImageFX system (Bio-  Fig.  2d. d, Chemical-shift-based secondary structure score of Ctc1 OB-A in the presence (yellow) and absence (gray) of p50 peptide. The scores are determined using TALOS+ (ref 44 ). Top and bottom edges of each bar represent the probabilities of each residue assigned to be α helix and β sheet, respectively. The secondary structure of Ctc1 OB-A observed in the cryo-EM structure is shown below for comparison. e, Plot of long range (greater than 5 residues) 1 H-1 H NOE restraints observed within Ctc1 OB-A. Residues with pairwise NOE restraint(s) are connected by a link. Thickness of each link is coded as indicated based on the number of NOE restraints between the two connected residues. Fig. 4: Identifying the "invisible" interface between Ctc1 OB-A and p50 peptide using NMR methods. a, Schematic diagram of p50 and constructs of p50 peptide. The N-terminal 30 kDa and 25 kDa fragments of p50 are labeled as p50N30 and p50N25, respectively. Previous biochemical study showed that p50N30 could bind Ctc1, whereas p50N25 could not 43 . The cryo-EM structure of p50 ends at residue 208 (Fig. 2b). Based on these facts, a series of p50 peptides in the range of residues 213-255 were designed to explore additional interface between p50 and Ctc1 OB-A that are "invisible" in the cryo-EM structure. b, NMR binding study of p50 peptides with Ctc1 OB-A. Two regions of 1 H-15 N HSQC spectra of 15 N-labelled Ctc1 OB-A in the absence (apo) and presence of unlabeled p50 peptides were shown. Chemical shifts of T71 and A73 were chosen to illustrate the binding process in this and the following panels c and d. p50228-250 peptide is determined to be the optimal construct and was used for other NMR studies presented in this paper. c, Titration series of p50 peptide into 15 N-labelled Ctc1 OB-A. The binding is in the slow exchange regime and saturated at 1:1 stoichiometry. d, Truncations of two unstructured loops (residues 38-49 and 131-146) of Ctc1 OB-A individually have no effect on its binding with p50 peptide. e, Secondary structure score of p50228-250 in the presence of Ctc1 OB-A. f, CSP index of p50 peptide upon binding Ctc1 OB-A. 1 H-15 N HSQC spectra shown in Fig. 2c were used for the CSP calculation. g, Model of the interactions between Ctc1 OB-A and p50. CS-Rosetta models of p50228-250 are shown in gray box with arrows pointing to the binding surface on Ctc1 OB-A. Unstructured linkers are shown as dashed lines. The composite map is generated using Phenix 70 with two focused refined maps (detailed in methods). g, Representative cryo-EM densities (gray mesh) encasing the related atomic models (colored sticks and ribbons).

Extended Data
longer than those in human and yeast (d). The α19 is structured only in Tt POLA1 when binding TtCST (e). f, Zoom-in views of the interface between Tt POLA1core α19 (ribbon) and TtCST (surface/electrostatic surface). In the lower panel, locations of positively and negatively charged residues on α19 are indicated using blue and red balls, respectively. g, Sequence conservation analysis of the β11-β12 hairpin and α19 of POLA1. Charged residues on α19 are indicated with black arrows. h, Zoom-in view of the interface between POLA1core and TtCST with sstDNA. Cryo-EM density are shown as transparent surface. The template entry port formed by POLA1core NTD and Exo and Ctc1 OB-C is indicated by a cycle. i, Path of sstDNA in the cryo-EM structure of Tt telomerase-PolαPrim. The sstDNA binds in the C-shape cleft of Ctc1 OB-C with its 5' side, while its 3' side passes through the template entry port to the active site of POLA1core (right). A G-quadruplex (GQ) formed by four Tt telomere repeats (modeled based on PDB 7JKU) is observed on a positively charged DNA binding surface of POLA1core between the palm and thumb (middle). j, Superimposition of the GQ structure and cryo-EM density. Weak density of sstDNA can be observed connecting the sstDNA on Ctc1 OB-C to the GQ.