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Massively parallel determination and modeling of endonuclease substrate specificity
We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucle...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267613/ https://www.ncbi.nlm.nih.gov/pubmed/25389263 http://dx.doi.org/10.1093/nar/gku1096 |
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author | Thyme, Summer B. Song, Yifan Brunette, T. J. Szeto, Mindy D. Kusak, Lara Bradley, Philip Baker, David |
author_facet | Thyme, Summer B. Song, Yifan Brunette, T. J. Szeto, Mindy D. Kusak, Lara Bradley, Philip Baker, David |
author_sort | Thyme, Summer B. |
collection | PubMed |
description | We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution. |
format | Online Article Text |
id | pubmed-4267613 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-42676132014-12-23 Massively parallel determination and modeling of endonuclease substrate specificity Thyme, Summer B. Song, Yifan Brunette, T. J. Szeto, Mindy D. Kusak, Lara Bradley, Philip Baker, David Nucleic Acids Res Nucleic Acid Enzymes We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution. Oxford University Press 2014-12-16 2014-11-11 /pmc/articles/PMC4267613/ /pubmed/25389263 http://dx.doi.org/10.1093/nar/gku1096 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Nucleic Acid Enzymes Thyme, Summer B. Song, Yifan Brunette, T. J. Szeto, Mindy D. Kusak, Lara Bradley, Philip Baker, David Massively parallel determination and modeling of endonuclease substrate specificity |
title | Massively parallel determination and modeling of endonuclease substrate specificity |
title_full | Massively parallel determination and modeling of endonuclease substrate specificity |
title_fullStr | Massively parallel determination and modeling of endonuclease substrate specificity |
title_full_unstemmed | Massively parallel determination and modeling of endonuclease substrate specificity |
title_short | Massively parallel determination and modeling of endonuclease substrate specificity |
title_sort | massively parallel determination and modeling of endonuclease substrate specificity |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267613/ https://www.ncbi.nlm.nih.gov/pubmed/25389263 http://dx.doi.org/10.1093/nar/gku1096 |
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