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Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein

Lisitskaya, Lidia and Petushkov, Ivan and Esyunina, Daria and Aravin, Alexei A. and Kulbachinskiy, Andrey (2020) Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein. Biochemical and Biophysical Research Communications, 533 (4). pp. 1484-1489. ISSN 0006-291X. https://resolver.caltech.edu/CaltechAUTHORS:20201027-090926979

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Abstract

In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.bbrc.2020.10.051DOIArticle
ORCID:
AuthorORCID
Lisitskaya, Lidia0000-0002-5283-7983
Esyunina, Daria0000-0002-3706-4425
Aravin, Alexei A.0000-0002-6956-8257
Kulbachinskiy, Andrey0000-0002-2292-6424
Additional Information:© 2020 Elsevier Inc. Received 15 October 2020, Accepted 18 October 2020, Available online 24 October 2020. This work was supported in part by the Russian Science Foundation (19-14-00359; analysis of ternary complex formation) and the Russian Foundation for Basic Research (18-29-07086; footprinting experiments). The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funders:
Funding AgencyGrant Number
Russian Science Foundation19-14-00359
Russian Foundation for Basic Research18-29-07086
Subject Keywords:Argonaute proteins; pAgo; Small RNA; Small DNA; DNA targeting
Issue or Number:4
Record Number:CaltechAUTHORS:20201027-090926979
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201027-090926979
Official Citation:Lidia Lisitskaya, Ivan Petushkov, Daria Esyunina, Alexei Aravin, Andrey Kulbachinskiy, Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein, Biochemical and Biophysical Research Communications, Volume 533, Issue 4, 2020, Pages 1484-1489, ISSN 0006-291X, https://doi.org/10.1016/j.bbrc.2020.10.051. (http://www.sciencedirect.com/science/article/pii/S0006291X20319690)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106294
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:27 Oct 2020 18:36
Last Modified:11 Dec 2020 22:11

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