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. 2013 Jun 24;8(6):e66756.
doi: 10.1371/journal.pone.0066756. Print 2013.

Structure of the Human Telomeric Stn1-Ten1 Capping Complex

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Free PMC article

Structure of the Human Telomeric Stn1-Ten1 Capping Complex

Christopher Bryan et al. PLoS One. .
Free PMC article

Abstract

The identification of the human homologue of the yeast CST in 2009 posed a new challenge in our understanding of the mechanism of telomere capping in higher eukaryotes. The high-resolution structure of the human Stn1-Ten1 (hStn1-Ten1) complex presented here reveals that hStn1 consists of an OB domain and tandem C-terminal wHTH motifs, while hTen1 consists of a single OB fold. Contacts between the OB domains facilitate formation of a complex that is strikingly similar to the replication protein A (RPA) and yeast Stn1-Ten1 (Ten1) complexes. The hStn1-Ten1 complex exhibits non-specific single-stranded DNA activity that is primarily dependent on hStn1. Cells expressing hStn1 mutants defective for dimerization with hTen1 display elongated telomeres and telomere defects associated with telomere uncapping, suggesting that the telomeric function of hCST is hTen1 dependent. Taken together the data presented here show that the structure of the hStn1-Ten1 subcomplex is conserved across species. Cell based assays indicate that hTen1 is critical for the telomeric function of hCST, both in telomere protection and downregulation of telomerase function.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Crystal structures of hStn1 and hTen1.
(A) The N-terminal domain of hStn1, (B) full-length hTen1, and (C) The C-terminal domain of hStn1.
Figure 2
Figure 2. Structure of the hStn1N-Ten1 heterodimer.
(A) Primary structures of hStn1 and hTen1 showing interacting domains. (B) Crystal structure of the hStn1-Ten1 dimer in cartoon representation looking down the interface of the two subunits. (C) View of the hStn1-Ten1 dimer rotated 180° to highlight the arrangement of OB fold putative DNA binding pockets. (D) Dimerization contacts between the hStn1N and full-length hTen1 C-terminal helices α3 and α2 respectively. Residues mutated in this study for ITC and cell based assays are shown in boxes. (E) Dimerization contacts between the β-barrels of hStn1N and hTen1.
Figure 3
Figure 3. DNA binding properties of hStn1, hTen1 and the hStn1-Ten1 complex.
Fluorescence Polarization (FP) data for (A) hStn1 (B) hTen1 and (C) the full length hStn1-Ten1 complex with ssDNA of various lengths (tel8, tel12, tel18, rand18, tel30– Table 3) (D) Table of hStn1, hTen1 and full-length hStn1-Ten1, ssDNA dissociation constants (Kd) calculated from the FP data of panels A, B and C.
Figure 4
Figure 4. hStn1 and hTen1 surface aminoacid conservation.
(A) Tertiary structure (cartoon) of hTen1 highlighting the putative DNA binding pocket (PBP) with a dashed circle. (B and C) Conservation map for the PBP and the hStn1-interacting surface of hTen1 respectively. Blue indicates residue conservation and red, residues that are variable. (D) Tertiary structure (cartoon) of hStn1 highlighting the PBP with a dashed circle. (E and F) Conservation map for the putative binding pocket of hStn1 and the hTen1-interacting surface respectively.
Figure 5
Figure 5. Isothermal titration calorimetry (ITC) data of hStn1 and hTen1 association.
(A) hStn1(WT) with hTen1(WT). (B) hStn1(WT) with hTen1(R27Q). (C) hStn1(WT) with hTen1(Y115A). (D) hStn1(WT) with hTen1(R119Q). (E) hStn1(D78A/I164A) with hTen1(WT). (F) hStn1(D78A/M167A) with hTen1(WT). (G) Table of ITC values for the full length, WT, single and double mutant hStn1 and hTen1 proteins obtained from the curve fit of figures 6A–F.
Figure 6
Figure 6. Southern blots analysis of telomeric DNA from cells carrying wild type (WT) or mutant hStn1 defective of hTen1 binding.
(A) The gel shows telomere length at passages 4, 8 and 12 of cells carrying siGFP (Mock), hStn1-Rescue with WT protein, hStn1-KD (shRNA-S2) and hTen1-KD (shRNA-T1). The hStn1-KD (shRNA-S2) and double mutants defective of hTen1 binding show increased telomere length, compared to the siGFP (Mock). (B) The gel shows telomere length at passages 6, 7–9 and 12 of cells carrying siGFP (Mock), hStn1-KD (shRNA-S2) and the double mutants hStn1(D78A/I164A) and hStn1(D78A/M167A).
Figure 7
Figure 7. Fluorescence in situ hybridization (FISH) data of HEK 293 cells infected with: siGFP (Mock), hStn1-KD (shRNA-S2), hTen1-KD (shRNA-T1), hStn1-Rescue and hStn1 double mutants, hStn1(D78A/I164A) and hStn1(D78A/M167A),defective of hTen1 binding.
(A) FISH of chromosomes in mock-treated (siGFP) cells display normal telomeres. (B) and (C) Telomere defects observed in hStn1 knockdown (hStn1-KD/shRNA-S2) and the double mutant hStn1(D78A/I164A) that disrupts hStn1-Ten1 association. Green arrows point to fragile telomeres, and pink to telomere free ends. (D) Bar graph showing the levels of telomere free ends observed in siGFP(mock), hStn1-Rescue and hStn1(D78A/I164A) or hStn1(D78a/M167A) double mutants defective of hTen1 binding. (E) Bar graph showing the levels of fragile telomeres observed in siGFP(mock), hStn1-Rescue and hStn1(D78A/I164A) or hStn1(D78a/M167A) double mutants and mutant hStn1 defective of hTen1 binding. Error bars show the standard deviation from 3 independent experiments. An average of ∼1000 chromosomes were counted in each experiment.

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