The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization

Sealey, David C.F.; Zheng, Lirong; Taboski, Michael A.S.; Cruickshank, Jennifer; Ikura, Mitsuhiko; Harrington, Lea

doi:10.1093/nar/gkp1160

Cited by 49 publications

(66 citation statements)

References 89 publications

(155 reference statements)

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“…The requirement of residue R151 for interaction in our assay suggests that we are monitoring a different binding activity. We were unable to obtain a DNA binding affinity for the TEN domain using fluorescence anisotropy (data not shown), which is similar to results reported for both the Tetrahymena and human TEN domains using filter binding and electrophoretic mobility shift assays (Jacobs et al, 2006;Moriarty et al, 2005;Sealey et al, 2010). This result is not unexpected because a weak or rapid exchange between the DNA and the anchor-site would aid in translocation of the DNA substrate during telomeric synthesis.…”

Section: E76ksupporting

confidence: 67%

“…The anchor-site of human TERT has been shown to bind directly to a telomeric oligonucleotide through its N-terminal TEN domain in the absence of the RNA (Sealey et al, 2010;Wyatt et al, 2007;Wyatt et al, 2009). We developed a similar assay for the yeast TEN domain and found that MBP-Est2p TEN associates in a sequence-specific manner with telomeric DNA.…”

Section: E76kmentioning

confidence: 99%

“…Mutations within the Tetrahymena TEN domain decrease interaction with the DNA primer (Jacobs et al, 2006) and the primer can be photocrosslinked to a fragment containing the Tetrahymena and S. cerevisiae TEN domains (Lue, 2005;Romi et al, 2007). Direct binding assays have demonstrated that the isolated TEN domain from human and Tetrahymena binds telomeric DNA (Finger and Bryan, 2008;Sealey et al, 2010;Wyatt et al, 2007;Wyatt et al, 2009). In S. cerevisiae, a fragment of Est2p containing the TEN domain interacts with full-length TLC1 RNA and increasing amounts of DNA compete with the RNA for protein binding in vitro (Xia et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Bairley

Guillaume

Vega

et al. 2011

Journal of Cell Science

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SummaryTelomerase is a ribonucleoprotein complex that is required for maintenance of linear chromosome ends (telomeres). In yeast, the Est2 protein reverse transcribes a short template region of the TLC1 RNA using the chromosome terminus to prime replication. Yeast telomeres contain heterogeneous G 1-3 T sequences that arise from incomplete reverse transcription of the TLC1 template and alignment of the DNA primer at multiple sites within the template region. We have previously described mutations in the essential N-terminal TEN domain of Est2p that alter telomere sequences. Here, we demonstrate that one of these mutants, glutamic acid 76 to lysine (est2-LT E76K ), restricts possible alignments between the DNA primer and the TLC1 template. In addition, this mutant exhibits increased processivity in vivo. Within the context of the telomerase enzyme, the Est2p TEN domain is thought to contribute to enzyme processivity by mediating an anchor-site interaction with the DNA primer. We show that binding of the purified TEN domain (residues 1-161) to telomeric DNA is enhanced by the E76K mutation. These results support the idea that the anchor-site interaction contributes to telomerase processivity and suggest a role for the anchor site of yeast telomerase in mediating primer-template alignment within the active site.

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Section: E76ksupporting

confidence: 67%

Section: E76kmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Bairley

Guillaume

Vega

et al. 2011

Journal of Cell Science

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“…By synthesizing the repetitive telomeric sequence using its RNA template, telomerase prevents cellular senescence in somatic cells (4). Moreover, human telomerase reverse transcriptase (hTERT), as the rate-limiting step in the activation of telomerase, is known to be an accurate measure of telomerase activity (TA).…”

Section: Introductionmentioning

confidence: 99%

Prognostic role of telomerase activity in gastric adenocarcinoma: A meta-analysis

Lü

Deng²,

Cao

et al. 2012

Experimental and Therapeutic Medicine

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Abstract. Activation of telomerase is involved in carcinogenesis in most types of cancers. However, the prognostic value of telomerase activity (TA) in patients with gastric carcinoma (GC) remains controversial. We conducted a meta-analysis to assess the relationship between TA and the clinical outcome of GC. A meta-analysis of 18 studies (886 patients) was performed to evaluate the association between TA and metastasis-related parameters in GC patients by searching databases, including PubMed, MEDLINE, EMBASE, Web of Science databases, Cochrane Library and the Chinese Biomedical Literature database (CBM) (last search updated in October 2011). We used the odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association between TA and metastasis of GC. Our analysis results indicated that high telomerase activity expression tended to be associated with the presence of lymph node metastasis (866 patients) (OR=2.03, 95% CI 1.21-3.39, p=0.007), the depth of invasion (886 patients) (OR=1.87, 95% CI 1.30-2.70, p=0.0007), distant metastasis (407 patients) (OR=2.71, 95% CI 1.59-4.63, p=0.0002), tumor size (466 patients) (OR=2.14, 95% CI 1.31-3.50, p=0.002) and TNM stage (711 patients) (OR=2.39, 95% CI 1.30-4.41, p=0.005). However, high TA expression was not associated with the presence of histologic differentiation (791 patients) (OR=1.51, 95% CI 0.73-3.11, p=0.26). In conclusion, telomerase overexpression not only plays a key role in primary initiation, but also promotes invasion and metastatic progression of GC.These findings raise the possibility of using TA to screen for the prognosis of gastric cancer.

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“…In addition, mouse (m)TERT levels also appear to diminish over time in primary cell cultures, in part, leading to cell senescence and cell death (1,6,10,14,18). Work with human primary cell lines has shown that ectopic expression of human TERT (hTERT) can lead to continued cell replication and hence, immortalization (21,31,37,39). More importantly, recent reports have shown immortalized hTERT cells remain differentiated, thus preserving the phenotypic properties that closely resemble in vivo characteristics (37,39).…”

Section: Epithelial; Polycysticmentioning

confidence: 99%

Telomerase immortalization of principal cells from mouse collecting duct

Steele¹,

Kolb

et al. 2010

American Journal of Physiology-Renal Physiology

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Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

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The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization

Cited by 49 publications

References 89 publications

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Prognostic role of telomerase activity in gastric adenocarcinoma: A meta-analysis

Telomerase immortalization of principal cells from mouse collecting duct

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