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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...

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Detalles Bibliográficos
Autores principales: Thyme, Summer B., Song, Yifan, Brunette, T. J., Szeto, Mindy D., Kusak, Lara, Bradley, Philip, Baker, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
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.
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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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