Nuclease dead Cas9 is a programmable roadblock for DNA replication

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities o...

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Autores principales: Whinn, Kelsey S., Kaur, Gurleen, Lewis, Jacob S., Schauer, Grant D., Mueller, Stefan H., Jergic, Slobodan, Maynard, Hamish, Gan, Zhong Yan, Naganbabu, Matharishwan, Bruchez, Marcel P., O’Donnell, Michael E., Dixon, Nicholas E., van Oijen, Antoine M., Ghodke, Harshad
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746809/
https://www.ncbi.nlm.nih.gov/pubmed/31527759
http://dx.doi.org/10.1038/s41598-019-49837-z
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author Whinn, Kelsey S.
Kaur, Gurleen
Lewis, Jacob S.
Schauer, Grant D.
Mueller, Stefan H.
Jergic, Slobodan
Maynard, Hamish
Gan, Zhong Yan
Naganbabu, Matharishwan
Bruchez, Marcel P.
O’Donnell, Michael E.
Dixon, Nicholas E.
van Oijen, Antoine M.
Ghodke, Harshad
author_facet Whinn, Kelsey S.
Kaur, Gurleen
Lewis, Jacob S.
Schauer, Grant D.
Mueller, Stefan H.
Jergic, Slobodan
Maynard, Hamish
Gan, Zhong Yan
Naganbabu, Matharishwan
Bruchez, Marcel P.
O’Donnell, Michael E.
Dixon, Nicholas E.
van Oijen, Antoine M.
Ghodke, Harshad
author_sort Whinn, Kelsey S.
collection PubMed
description Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.
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spelling pubmed-67468092019-09-27 Nuclease dead Cas9 is a programmable roadblock for DNA replication Whinn, Kelsey S. Kaur, Gurleen Lewis, Jacob S. Schauer, Grant D. Mueller, Stefan H. Jergic, Slobodan Maynard, Hamish Gan, Zhong Yan Naganbabu, Matharishwan Bruchez, Marcel P. O’Donnell, Michael E. Dixon, Nicholas E. van Oijen, Antoine M. Ghodke, Harshad Sci Rep Article Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro. Nature Publishing Group UK 2019-09-16 /pmc/articles/PMC6746809/ /pubmed/31527759 http://dx.doi.org/10.1038/s41598-019-49837-z Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Whinn, Kelsey S.
Kaur, Gurleen
Lewis, Jacob S.
Schauer, Grant D.
Mueller, Stefan H.
Jergic, Slobodan
Maynard, Hamish
Gan, Zhong Yan
Naganbabu, Matharishwan
Bruchez, Marcel P.
O’Donnell, Michael E.
Dixon, Nicholas E.
van Oijen, Antoine M.
Ghodke, Harshad
Nuclease dead Cas9 is a programmable roadblock for DNA replication
title Nuclease dead Cas9 is a programmable roadblock for DNA replication
title_full Nuclease dead Cas9 is a programmable roadblock for DNA replication
title_fullStr Nuclease dead Cas9 is a programmable roadblock for DNA replication
title_full_unstemmed Nuclease dead Cas9 is a programmable roadblock for DNA replication
title_short Nuclease dead Cas9 is a programmable roadblock for DNA replication
title_sort nuclease dead cas9 is a programmable roadblock for dna replication
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746809/
https://www.ncbi.nlm.nih.gov/pubmed/31527759
http://dx.doi.org/10.1038/s41598-019-49837-z
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