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The telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase.

ABSTRACT: The central hallmark of telomerases is repetitive copying of a short, defined sequence within its integral RNA subunit. We sought to identify structural determinants of this unique activity in the catalytic protein subunit telomerase reverse transcriptase (TERT) of telomerase. Residues within the highly conserved telomerase-specific T motif of human TERT were mutationally probed, leading to variant telomerases with increased repeat extension rates and wild-type processivity. The extension rate increases were independent of template sequence composition and only moderately correlated to telomerase RNA (TR) binding. Importantly, analysis of substrate primer elongation showed that the extension rate increases primarily resulted from increases in the repeat (type II) translocation rate. Our findings indicate a participatory role for the T motif in repeat translocation, an obligatory event for repetitive telomeric DNA synthesis. Thus, the T motif serves as a restrictive determinant of repetitive reverse transcription.

SUBMITTER: Drosopoulos WC

PROVIDER: S-EPMC2798474 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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The telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase.

Drosopoulos William C WC Prasad Vinayaka R VR

Molecular and cellular biology 20091116 2

The central hallmark of telomerases is repetitive copying of a short, defined sequence within its integral RNA subunit. We sought to identify structural determinants of this unique activity in the catalytic protein subunit telomerase reverse transcriptase (TERT) of telomerase. Residues within the highly conserved telomerase-specific T motif of human TERT were mutationally probed, leading to variant telomerases with increased repeat extension rates and wild-type processivity. The extension rate i ...[more]

PMID: 19917726

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Project description:The aims of this study were to generate periodontal ligament (PDL) cells that have adenovirus- or lentivirus-mediated overexpression of human telomerase reverse transcriptase (hTERT) and to compare the osteogenic and proliferative abilities of the two cell lines to establish an efficient and stable cell model that will be more suitable for studies of PDL regeneration. After construction of the recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT, human PDL cells were infected by packaged adenovirus and lentivirus particles to establish two PDL cell lines. The expression levels of hTERT and mRNA for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, core-binding factor (runt-related transcription factor 2), and type I collagen were assessed for each cell line. After culture in osteoinductive culture medium for 14 days, the PDL cells were stained with alizarin red to observe formation of mineralized nodules, and proliferation activity was measured with a CCK-8 kit. A quantitative polymerase chain reaction assay indicated that the two transduced cell lines expressed hTERT levels that were significantly higher than that seen for normal PDL cells. Expression of all osteogenic genes tested, with the exception of osteopontin, was higher for both the adenovirus- and lentivirus-transduced cells relative to normal PDL cells. The expression of bone sialoprotein, osteocalcin, and runt-related transcription factor 2 in adenovirus-transduced cells was significantly higher than that for lentivirus-transduced cells. Alizarin red staining showed that the adenovirus-transduced cell line produced more mineralized nodules than the lentivirus-transduced cell line, whereas a CCK-8 test showed that the adenovirus-transduced cell line had higher proliferation activity than lentivirus-transduced cells. In conclusion, a PDL cell line established by adenovirus transduction had superior osteogenic differentiation and proliferative activity compared to the cell line produced by lentivirus transduction. The results indicate that PDL cells having adenovirus-mediated expression of hTERT would be a more suitable model for studies of PDL regeneration.

Dataset Information

The telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase.

Publications

The telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase.

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