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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Detalles Bibliográficos
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
Descripción
Sumario: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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