Extended Data Fig. 9: Comparison of TtCST and human CST.
From: Structure of Tetrahymena telomerase-bound CST with polymerase α-primase
a, Domain diagrams of TtCST and human CST. b, Structural homology analysis of individual OB domains of TtCtc1 (OB-A to -C) and human CTC1 (OB-A to -G) using the Dali sever70. On the basis of the resulted pairwise Z-scores, TtCtc1 OB-B and OB-C are identified as homologues of human CTC1 OB-F and OB-G, respectively. c, Structural comparison of individual domains from TtCST (colour) with corresponding domains from human CST (grey). Structures of WH-WH domains of Tt Stn1 and human STN1 were superposed based on WH1 or WH2 domain. The relative orientation of the two WH domains is different between Tt and human. d, The interface between Stn1 WH-WH and Ctc1 in the cryo-EM structure of Tt telomerase–PolαPrim. Cryo-EM densities are shown as transparent surfaces. An previously unstructured loop of Ctc1 OB-C (Extended Data Fig. 2b) partially forms an α helix (α520-540) and contributes to the interface with Stn1 WH-WH. e, Comparison of DNA binding sites on TtCtc1 (colour) and human CTC1 (grey). Conserved residues located on the DNA binding interface are shown as sticks. Cryo-EM densities of TtCtc1 are shown as transparent surfaces. In the decameric structure of human CST19 (PDB 6W6W), sstDNA primarily binds on CTC1 OB-F. However, in TtCST, the equivalent sstDNA binding site on OB-B is partially occluded by a helix (α6) that is part of an unstructured loop in hCTC1 OB-F. The helix α6 abuts TERT in TtCST without PolαPrim (Extended Data Fig. 2k) and Stn1 WH2 when PolαPrim is bound (as shown in d). f, Sequence conservation analysis of Ctc1 residues on the DNA binding interface. Residues shown in e are indicated with black arrows. Conserved cysteines in the zinc ribbon motifs are indicated with pink arrows. g, SEC profile and SDS-PAGE gel of TtCST–p50 co-expressed in Sf9 cells. Gel samples are from the peak fractions of the SEC profile as indicated. h, EMSA of purified wild-type TtCST with d(GTTGGG)n, where n = 3, 5 or 10. i, Substitutions of TtCtc1 OB-B conserved residues K303E/K306E/F308A and F264A/Y268A substantially decrease d(GTTGGG)5 binding, as indicated by EMSAs. These results suggest that the binding site on TtCtc1 OB-B might be accessible to sstDNA in free TtCST where neither TERT nor Stn1 WH-WH stabilize helix α6. Wedges indicate two-fold dilution of TtCST. The first lane of each gel is a TtCST-free control. j, Quantifications of fraction of bound DNA for all the independent EMSA experiments with TtCST WT and variants as indicated (n = 3 biological replicates). KD values were determined as described in Methods. k, Effect of TtCST residue substitutions on d(GTTGGG)5 binding. Data are mean ± s.d. from n = 3 biological replicates shown in j. *P = 0.04, **P = 0.009, ****P < 0.0001; one-tailed unpaired t-tests.
