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Vol. 16, Issue 7, 3152-3161, July 2005

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

Tara J. Moriarty ^* , Ryan J. Ward  , Michael A.S. Taboski ^*  , and Chantal Autexier ^*  

^* Department of Anatomy and Cell Biology, Experimental Medicine Division, McGill University, Montréal, Québec H3A 2B2, Canada; Department of Medicine, Experimental Medicine Division, McGill University, Montréal, Québec H3A 2B2, Canada; and Lady Davis Institute for Medical Research, Montréal, Québec H3T 1E2, Canada

Submitted February 23, 2005; Accepted April 18, 2005
Monitoring Editor: Joseph Gall

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.

Abbreviations used: TERT, telomerase reverse transcriptase; RID1, RNA interaction domain 1; DAT, dissociates activities of telomerase; TR, telomerase RNA; nt, nucleotide; WT, wild-type; TRAP, telomeric repeat amplification protocol; RRL, rabbit reticulocyte lysate.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.

Address correspondence to: Chantal Autexier (chantal.autexier{at}mcgill.ca).

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