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. 2012 Dec 13;492(7428):285-9.
doi: 10.1038/nature11648. Epub 2012 Oct 24.

The TEL Patch of Telomere Protein TPP1 Mediates Telomerase Recruitment and Processivity

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Free PMC article

The TEL Patch of Telomere Protein TPP1 Mediates Telomerase Recruitment and Processivity

Jayakrishnan Nandakumar et al. Nature. .
Free PMC article

Abstract

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event). Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15-17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.

Figures

Figure 1
Figure 1. Separation-of-function mutants of TPP1 affect telomerase processivity without affecting telomere complex formation
a, Domain architecture of POT1-TPP1 shown alongside the crystal structures of the DNA-binding domain of POT1 (cyan surface) bound to DNA (red ribbon; PDB: 1XJV) and the TPP1-OB domain (PDB: 2I46). b, DNA binding curves from filter-binding of mixtures containing trace amounts of 32P-labeled ssDNA (GGTTAGGGTTAG), 200 nM TPP1, and varying concentrations of POT1. See Supp. Fig. 2a for KD values. c, Direct telomerase activity assay with primer a5 of lysates from cells co-transfected with a TR plasmid and FLAG-tagged POT1, TPP1, and TERT plasmids. “No POT1-TPP1”, transfection without POT1 and TPP1. (Left), number of telomeric repeats added to primer. d, Processive extension (>15 repeats/ total) with TPP1 mutants relative to that with WT TPP1 (green line) obtained from three independent sets of experiments (as in panel c); error bars, standard deviations. Stimulation of processivity is assessed relative to the “No POT1-TPP1” negative control (red line). Two-tailed Student t-test with respect to R175V: *P<0.02, **P<0.005. Red label: significantly defective; green label: not significantly defective. e, Immunoblot of lysates used in panel c probed with anti-FLAG antibody-HRP conjugate shows uniform expression.
Figure 2
Figure 2. TPP1 mutations that disrupt telomerase stimulation also disrupt telomerase binding
a, Pull-down of transiently expressed FLAG-TPP1, FLAG-POT1 and associated untagged TERT from HeLa-EM2-11ht lysates on anti-FLAG conjugated beads. Input, immunoblot of soluble cellular lysates prior to incubation with beads. Beads, immunoblot of proteins retained on antibody beads after 2 h incubation at 4°C and washing. The TERT signal was plotted after correction for loading differences (using the actin signal). b, Comparison of TERT pull-down by FLAG-TPP1 mutants in the presence of FLAG-POT1 and primer a5. Control 1, FLAG-POT1/TPP1 and primer a5 omitted. Control 2, FLAG-TPP1 (WT) present, FLAG-POT1 and primer omitted. Control 3, FLAG-POT1 and primer present, FLAG-TPP1 omitted. c, The mean TERT signal on beads obtained from quantification of three independent sets of experiments of which panel b is representative; error bars, standard deviations. Two-tailed Student t-test with respect to R175V: *P<0.05, **P<0.01. Red label: significantly defective; green label: not significantly defective. d, The TEL patch (amino acids in orange), the surface of the OB domain of TPP1 that mediates telomerase association and stimulation. Other amino acids mutated in this study (cyan).
Figure 3
Figure 3. TPP1 TEL-patch mutants fail to stimulate telomere lengthening in human cells
a, Engineering HeLa-EM2-11ht stable cell lines containing single-copy integration of bidirectional Tet-inducible shTPP1 and shRNA-resistant FLAG-TPP1* (WT or mutants) genes. b, A stable cell line encoding shTPP1 (no exogenous FLAG-TPP1) shows doxycycline-dependent knockdown of endogenous TPP1 protein. Non-specific bands serve as loading controls. c, Quantitative RT-PCR showing knockdown of endogenous TPP1 mRNA in the indicated cell lines ± S.D. (n =3). d, Western blot showing similar protein levels of shRNA-resistant FLAG-TPP1 in the indicated cell lines also expressing shTPP1. e, Telomeric restriction fragment (TRF) Southern blot of DNA from HeLa-EM2-11ht (Untransfected HeLa) and stable cell lines expressing shTPP1 and the indicated TPP1 constructs at the indicated population doublings (PD). Left lanes, DNA length standards. f, Change in mean telomere length for data shown in panel e was plotted against PD. The vertical bars at the right indicate the distinct ranges of telomere length attained by WT* & R175V* versus E169A/E171A* & L212A* cells after 81 PD.
Figure 4
Figure 4. Failure to stimulate telomere lengthening correlates with inability to recruit telomerase to telomeres
a, Fluorescence in situ hybridization (FISH) detects TR (green) and immunofluorescence (IF) detects the indicated FLAG-TPP1 proteins (red). Merge, Yellow spots indicate recruitment of telomerase to telomeres. b, Quantification of telomerase recruitment data of which panel a is representative. The average “% FLAG-TPP1 foci that contain TR” and standard deviations (error bars) of 15 fields of view (40–120 cells total) were plotted for the indicated stable cell lines. c, FISH-IF experiment showing that telomerase (TR in green) in E169A/E171A* and L212A* cells fails to be recruited to telomeres (FLAG-TPP1 in red) and instead remains localized in Cajal bodies (coilin in blue).

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