POT1 recruits and regulates CST-Polα/Primase at human telomeres

Abstract

Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1-Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/Primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Phosphorylation of POT1 is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/Primase in an inactive auto-inhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/Primase into an active state that completes telomere replication through fill-in synthesis.

Keywords: CST; DNA replication; DNA-protein complex; POT1; Polymerase α/Primase; cryo-EM; phospho-regulation; shelterin; telomere.

Fig. 1.. POT1-CST interaction.

(A) Immunoblot of anti-Myc co-IPs of Myc-tagged POT1 proteins and FLAG-tagged CST from co-transfected 293T cells showing that mPOT1b interacts with human CST better than human POT1. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. (B) Design of constructs encoding chimeric swaps between mPOT1b (red) and human POT1 (salmon). HMb: Human/Mouse POT1b swap; OB: oligosaccharide/oligonucleotide-binding domain; HJRL: Holliday junction resolvase-like domain. (C) Immunoblots of CST binding by chimeric human-mouse POT1 proteins. Anti-Myc co-IPs of Myc-tagged POT1 constructs, and FLAG-tagged CST from co-transfected 293T cells showing that aa 323–326 of mPOT1b are important for the CST interaction (comparison of HMb4 and HMb5). Immunoblots were probed with anti-Myc and anti-FLAG antibodies. (D) Sequence alignment of a region in the POT1 hinge (aa 309–330) (left) and the effect of the sequence on the interaction with CST (right). HMb4 contains the POT1 hinge and HMb1 contains the mPOT1b hinge. HMb3 and HMb5 contain the same hinge sequence shown, but they represent different constructs as shown in (B). (E) Cartoon schematics and gels of purified POT1/TPP1 and CST proteins used for fluorescent size-exclusion chromatography (FSEC) analysis. (F-G) FSEC analysis of the interaction between CST and POT1(WT)/GFP-TPP1 (blue) or POT1(ESDL)/GFP-TPP1 (red) in the absence (F) or presence (G) of telomeric ssDNA. Traces without CST are shown as dashed lines and color key is the same for both panels. RFU: relative fluorescence units.

Fig. 2.. Cryo-EM structures of CST–POT1(ESDL)/TPP1 complexes.

(A) Domain organization of CST and the shelterin subunits POT1(ESDL) and TPP1. Regions not observed in the cryo-EM maps are shown at 50% opacity. Dashed lines indicate regions not included in the expression construct. *The TPP1 serine-rich linker was included in the apo complex but not the ssDNA-bound complex. ARODL: Acidic Rpa1 OB-binding Domain-like 3-helix bundle; wH: winged helix-turn-helix domain; RD: recruitment domain, TID: TIN2-interacting domain. For other abbreviations see Fig. 1. (B) Cryo-EM reconstruction of apo CST–POT1(ESDL)/TPP1 at 3.9-Å resolution (see also Fig. S3). (C) Cryo-EM reconstruction of ssDNA-bound CST–POT1(ESDL)/TPP1 at 4.3-Å resolution (see also Fig. S4). (D-E) Atomic models for the apo and ssDNA-bound CST–POT1(ESDL)/TPP1 complexes, respectively. See also Video S1.

Fig. 3.. Phosphorylation-dependent interaction of POT1(ESDL) with CST.

(A) Left panels: Structure of apo CST–POT1(ESDL)/TPP1 (left) and close-up view of the interaction of the POT1 hinge with Ctc1^OB-D (right). Right panels: Surface electrostatic potential analysis of the POT1(ESDL) hinge interacting with Ctc1^OB-D. The POT1(ESDL) hinge is shown in cartoon (left) and surface (right) representation with electrostatic potential colored from red (−10 k_BT/e) to white (0 k_BT/e) to blue (+10 k_BT/e) (see also Fig. S9). (B) Immunoblots of anti-Myc co-IPs of Myc-tagged POT1 constructs and FLAG-tagged CST from co-transfected 293T cells showing enhanced interaction with CST upon increasing negative charge of amino acids in the POT1 hinge. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. The sequences of the altered POT1 alleles in comparison to POT1 and mPOT1b are displayed above the immunoblot. The first three lanes also appear in Fig. 1A. They are reproduced here as the controls for the experiment, which was performed simultaneously. (C) FSEC analysis of the phosphorylation-dependent interaction between CST and POT1(WT)/GFP-TPP1 (top, blue), POT1(SED)/GFP-TPP1 (middle, purple), and POT1(ESDL)/GFP-TPP1 (bottom, red) in the presence (right) or absence (left) of telomeric ssDNA (Fig. S9B). Traces without CST are shown as dashed lines, and traces containing POT1/TPP1 that have been treated with λ protein phosphatase (λPP) are shown in orange. RFU: relative fluorescence units. The chromatograms for the WT and ESDL mock samples also appear in Fig. 1F-G. They are reproduced here as the controls for the phosphatase experiment, which was performed simultaneously.

Fig. 4.. CP mutations map to the primary Ctc1–POT1 interface.

(A) Comparison of CST-bound POT1^OB−3/TPP1 (this study, colored) to unbound POT1^OB−3/TPP1 (PDB 5H65/5UN7 ^,, grayscale). (B) Close-up views of sites of interest at or near the interface between POT1 and Ctc1 (see also Fig. S6). Residues colored in bright red are mutated in CP. (i) Self-interaction between POT1 Ser322 and Arg367. Salt bridges are shown as yellow dashed lines. CP mutation S322L is predicted to disrupt this salt bridge and affect phosphorylation of the hinge. (ii) Self-interaction between POT1 hinge and C-terminus. (iii) Localization of Gly503 in the hydrophobic core of Ctc1^ARODL predicts a destabilizing effect of the G503R CP mutation. (iv) Primary hydrophobic interface between POT1 hinge, POT1^OB−3, and Ctc1^ARODL. CP mutation H484P is predicted to disrupt the hydrophobic stacking interactions with POT1 Pro603 and Ctc1 His488 and Pro483. (v) Salt bridges between POT1 hinge residue Glu325 and Ctc1^OB-D residue Arg624.

Fig. 5.. POT1^OB−2 interacts with CST’s ssDNA-binding site, precluding formation of the CST–Polα/Primase PIC.

(A) Interaction between POT1^OB−2 and Ctc1 at the CST ssDNA-binding interface. (Left) The black box indicates the region of the apo CST–POT1(ESDL)/TPP1 model shown in the right panels. (Right) Zoom views of the interface. The first panel shows the ssDNA-bound CST structure (PDB 6W6W , gray with DNA colored). The second panel shows the same view of the CST–POT1(ESDL)/TPP1 structure (this study, colored). Ctc1 aa 909–927 are modeled as poly-alanine stubs, and no ssDNA is bound to Ctc1. The two structures are superposed in panel three. (B) Negative-stain EM 2D averages (left) of ssDNA-bound CST–POT1(ESDL)/TPP1 showing three major conformations depicted by the cartoon schematics (right). 2D averages are flipped or rotated by 90° increments from Fig. S4 into similar orientations for ease of comparison and are sorted by number of particles per class from the most populous class at top left to the least populous class at bottom right. The 2D averages that correspond to each cartoon state are indicated with black, gray, or white circles. The scale bar represents 333 Å (See also Fig. S4). (C) The CST–POT1(ESDL)/TPP1–ssDNA complex (left) and its superposition with the CST–Polα/Primase recruitment complex (RC, middle) and pre-initiation complex (PIC, right). The CST–POT1(ESDL)/TPP1–ssDNA complex is shown as an opaque surface, and CST–Polα/Primase complexes are shown in cartoon representation with transparent surfaces. Orthogonal views show that POT1(ESDL)/TPP1 binding does not obstruct the major interface of the RC, but POT1^OB−1/2 and Stn1^C would interfere with binding of the POLA1 catalytic core to the CST ssDNA-binding site in the PIC (see also Fig. S9). (D) Cartoon schematics showing how CST–POT1/TPP1 is incompatible with PIC formation but could form RC-like complexes in both monomeric and dimeric forms.

Fig. 6.. Model for telomere maintenance by shelterin and CST–Polα/Primase.

Following DNA replication, CST–Polα/Primase is recruited to telomeres in the auto-inhibited RC-like state by phosphorylated POT1 (1). The POT1–CST interaction prevents activation of CST–Polα/Primase during 5’ end resection (2) and telomerase action (3). After telomerase has elongated telomeres, a cell cycle-dependent switch results in dephosphorylation of POT1 and subsequent release of CST–Polα/Primase to the telomeric ssDNA (4). When released, CST–Polα/Primase can readily form the PIC and execute fill-in synthesis, thereby completing telomere replication to form fully functional G1 telomeres (5).