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Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

Kuzmenko, Anton and Yudin, Denis and Ryazansky, Sergei and Kulbachinskiy, Andrey and Aravin, Alexei A. (2019) Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea. Nucleic Acids Research, 47 (11). pp. 5822-5836. ISSN 0305-1048. PMCID PMC6582412. https://resolver.caltech.edu/CaltechAUTHORS:20190225-135750266

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Abstract

Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5′-nucleotide in guide DNA and can use not only 5′-phosphorylated but also 5′-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3′-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/nar/gkz379DOIArticle
https://doi.org/10.1101/558684DOIDiscussion Paper
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582412PubMed CentralArticle
ORCID:
AuthorORCID
Ryazansky, Sergei0000-0002-3171-2997
Kulbachinskiy, Andrey0000-0002-2292-6424
Alternate Title:Mesophilic pAgo nucleases for programmable DNA cleavage
Additional Information:© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial 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. Received: 21 February 2019; Revision Received: 27 April 2019; Accepted: 30 April 2019; Published: 22 May 2019. We thank A. Oguienko for assistance with figure preparation, D. Esyunina and M. Petrova for experimental support and helpful discussions. Funding: Ministry of Science and Higher Education of the Russian Federation [14.W03.31.0007]. Funding for open access charge: Ministry of Science and Higher Education of the Russian Federation [14.W03.31.0007]. Conflict of interest statement. None declared.
Funders:
Funding AgencyGrant Number
Ministry of Education and Science of the Russian Federation 14.W03.31.0007
Subject Keywords:Argonaute proteins, DNA cleavage, CbAgo, LrAgo, CRISPR-Cas
Issue or Number:11
PubMed Central ID:PMC6582412
Record Number:CaltechAUTHORS:20190225-135750266
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190225-135750266
Official Citation:Anton Kuzmenko, Denis Yudin, Sergei Ryazansky, Andrey Kulbachinskiy, Alexei A Aravin, Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea, Nucleic Acids Research, Volume 47, Issue 11, 20 June 2019, Pages 5822–5836, https://doi.org/10.1093/nar/gkz379
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:93230
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:25 Feb 2019 22:05
Last Modified:03 Oct 2019 20:52

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