title: 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.
authors.affiliation: Department of Anatomy and Cell Biology, Experimental Medicine Division, McGill University, Montréal, Québec H3A 2B2, Canada.
authors.forename: Tara J
authors.initials: TJ
authors.lastname: Moriarty
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Ward
authors.forename: Michael A S
authors.initials: MA
authors.lastname: Taboski
authors.forename: Chantal
authors.initials: C
authors.lastname: Autexier
mesh_terms: D020816:Amino Acid Motifs; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000648:ultrastructure; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004247:DNA; D019098:Telomerase