Moriarty Tara J, Ward Ryan J, Taboski Michael A S, Autexier Chantal
Department of Anatomy and Cell Biology, Experimental Medicine Division, McGill University, Montréal, Québec H3A 2B2, Canada.
Mol Biol Cell. 2005 Jul;16(7):3152-61. doi: 10.1091/mbc.e05-02-0148. Epub 2005 Apr 27.
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.
端粒酶介导的端粒DNA合成对于真核细胞的永生至关重要。端粒酶利用一种独特的持续合成重复序列的形式，将短的端粒重复序列片段添加到DNA底物上。有人提出，重复序列添加的持续性部分受端粒酶逆转录酶（TERT）依赖性锚定位点的调节；然而，在任何TERT中都尚未鉴定出介导锚定位点的残基。我们报告了一种N端人TERT（hTERT）RNA相互作用结构域1（RID1）突变的特征，该突变导致端粒酶活性缺陷，这与模板近端锚定位点的破坏一致，包括在短端粒引物上的持续性降低以及在具有非端粒5'序列的底物上的活性降低，但在具有富含G的非端粒5'序列的引物上则没有。该突变位于RID1的一个亚区域内，该亚区域先前与与催化活性无关的生物端粒酶功能有关（N-DAT结构域）。其他N-DAT和C端DAT（C-DAT）突变体以及C端标记的hTERT-HA变体在延长短端粒引物方面存在缺陷，并且通过增加DNA引物的浓度可以部分或完全挽救DAT变体的催化表型。这些观察结果表明，RID1和hTERT C端有助于端粒酶对其底物的亲和力，并且RID1可能构成人端粒酶锚定位点的一部分。
