An Anchor Site–Type Defect in Human Telomerase That Disrupts Telomere Length Maintenance and Cellular Immortalization

Abstract

Telomerase-mediated telomeric DNA synthesis is important for eukaryotic cell immortality. Telomerase adds tracts of short telomeric repeats to DNA substrates using a unique repeat addition form of processivity. It has been proposed that repeat addition processivity is partly regulated by a telomerase reverse transcriptase (TERT)-dependent anchor site; however, anchor site-mediating residues have not been identified in any TERT. We report the characterization of an N-terminal human TERT (hTERT) RNA interaction domain 1 (RID1) mutation that caused telomerase activity defects consistent with disruption of a template-proximal anchor site, including reduced processivity on short telomeric primers and reduced activity on substrates with nontelomeric 5' sequences, but not on primers with nontelomeric G-rich 5' sequences. This mutation was located within a subregion of RID1 previously implicated in biological telomerase functions unrelated to catalytic activity (N-DAT domain). Other N-DAT and C-terminal DAT (C-DAT) mutants and a C-terminally tagged hTERT-HA variant were defective in elongating short telomeric primers, and catalytic phenotypes of DAT variants were partially or completely rescued by increasing concentrations of DNA primers. These observations imply that RID1 and the hTERT C terminus contribute to telomerase's affinity for its substrate, and that RID1 may form part of the human telomerase anchor site.

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

“…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.…”

“…Assays of telomerase activity implicate an N-terminal domain of TERT [called telomerase essential N-terminus (TEN), GQ or Region I (Friedman and Cech, 1999;Jacobs et al, 2006;O'Connor et al, 2005;Xia et al, 2000)] in anchor-site function (Lue, 2004;Lue, 2005;Moriarty et al, 2005). 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).…”

Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

“…Furthermore, these results show that the N-DAT domain in mTERT, which is 82% similar to the human DAT region, functions properly in human cells (2). The N-DAT domain has been proposed to mediate telomerase access to telomeric chromatin (3,4) or to contribute to interactions between telomerase and telomere DNA repeats (40,52). Instead, our data identify the C terminus as a critical regulatory domain controlling the ability of TERT to stabilize telomeres in cells and to extend replicative lifespan.…”

“…These DAT (dissociates activities of telomerase) domains were proposed to be required for the recruitment of telomerase to telomeric chromatin (3,4). Other analyses of these TERT mutants found that the DAT mutants showed reduced processivity, principally on oligonucleotide primers matching the natural telomere sequence, indicating that the DAT regions contribute to interactions between TERT and its telomere substrate (40,52). These data suggest that the DAT mutants may fail to immortalize human cells because of impaired interaction with telomere DNA sequences or with telomeric chromatin.…”

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

“…Studies have demonstrated that the human telomerase enzyme is one of the most processive telomerase complexes, especially compared to the murine one [10,11]. Human telomerase processivity is mediated by template and nontemplate regions of human TR (hTR) and by regions in human TERT (hTERT), specifically the C-terminal and N-terminal RNA interaction domain 1 [12][13][14][15][16][17]. The ectopic expression of hTERT enables the immortalization not only of human primary cells, but also of other primary vertebrate cells such as leporine, bovine, porcine, simian and cervine cells [18][19][20][21][22][23].…”