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A single-molecule assay for telomerase structure-function analysis
The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule ass...
Autores principales: | , , |
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2010
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817460/ https://www.ncbi.nlm.nih.gov/pubmed/19920121 http://dx.doi.org/10.1093/nar/gkp1033 |
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author | Wu, John Y. Stone, Michael D. Zhuang, Xiaowei |
author_facet | Wu, John Y. Stone, Michael D. Zhuang, Xiaowei |
author_sort | Wu, John Y. |
collection | PubMed |
description | The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. |
format | Text |
id | pubmed-2817460 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-28174602010-02-08 A single-molecule assay for telomerase structure-function analysis Wu, John Y. Stone, Michael D. Zhuang, Xiaowei Nucleic Acids Res Methods Online The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. Oxford University Press 2010-01 2009-11-17 /pmc/articles/PMC2817460/ /pubmed/19920121 http://dx.doi.org/10.1093/nar/gkp1033 Text en © The Author(s) 2009. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Wu, John Y. Stone, Michael D. Zhuang, Xiaowei A single-molecule assay for telomerase structure-function analysis |
title | A single-molecule assay for telomerase structure-function analysis |
title_full | A single-molecule assay for telomerase structure-function analysis |
title_fullStr | A single-molecule assay for telomerase structure-function analysis |
title_full_unstemmed | A single-molecule assay for telomerase structure-function analysis |
title_short | A single-molecule assay for telomerase structure-function analysis |
title_sort | single-molecule assay for telomerase structure-function analysis |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817460/ https://www.ncbi.nlm.nih.gov/pubmed/19920121 http://dx.doi.org/10.1093/nar/gkp1033 |
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