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Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin
Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse t...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3300002/ https://www.ncbi.nlm.nih.gov/pubmed/22187156 http://dx.doi.org/10.1093/nar/gkr1243 |
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author | Gardano, Laura Holland, Linda Oulton, Rena Le Bihan, Thierry Harrington, Lea |
author_facet | Gardano, Laura Holland, Linda Oulton, Rena Le Bihan, Thierry Harrington, Lea |
author_sort | Gardano, Laura |
collection | PubMed |
description | Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. |
format | Online Article Text |
id | pubmed-3300002 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-33000022012-03-13 Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin Gardano, Laura Holland, Linda Oulton, Rena Le Bihan, Thierry Harrington, Lea Nucleic Acids Res Methods Online Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. Oxford University Press 2012-03 2011-12-19 /pmc/articles/PMC3300002/ /pubmed/22187156 http://dx.doi.org/10.1093/nar/gkr1243 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Gardano, Laura Holland, Linda Oulton, Rena Le Bihan, Thierry Harrington, Lea Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title | Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title_full | Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title_fullStr | Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title_full_unstemmed | Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title_short | Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
title_sort | native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3300002/ https://www.ncbi.nlm.nih.gov/pubmed/22187156 http://dx.doi.org/10.1093/nar/gkr1243 |
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