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Analysis of a putative nuclease associated with the Argonaute protein from Rhodobacter sphaeroides

Lisitskaya, L. and Esyunina, D. and Komar, A. and Aravin, A. and Kulbachinskiy, A. (2021) Analysis of a putative nuclease associated with the Argonaute protein from Rhodobacter sphaeroides. FEBS Open Bio, 11 (S1). p. 238. ISSN 2211-5463. doi:10.1002/2211-5463.13205.

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The Argonaute protein from the alphaproteobacterium Rhodobacter sphaeroides (RsAgo) binds small guide RNAs to recognize complementary DNA targets. In bacterial cells, RsAgo is bound to both small RNAs and small DNAs, with a preference for foreign genetic elements. RsAgo also promotes degradation of plasmid DNA in vivo. However, RsAgo itself lacks the slicer activity, suggesting the involvement of additional nucleases. To shed light on the molecular mechanism of target processing, we study a putative nuclease which is located in the same operon with RsAgo in the R. sphaeroides genome. This protein has motifs characteristic for the PD(D/E)XK superfamily of nucleases. Numerous attempts to express the wildtype nuclease gene in Escherichia coli were not successful due to a low level of expression and low solubility of the recombinant protein. A codon-optimized sequence of the nuclease gene was cloned and sucessfully overexpressed in E. coli. Different expression vectors were used to produce histidine-tagged nuclease and a fusion protein with a chitin-binding domain, which allowed to obtain highly pure nuclease preparations. Initial assays did not reveal nuclease activity in these proteins. However, it was shown that coexpression of nuclease with the RsAgo protein in E. coli significantly increases the yield of RsAgo and changes the spectrum of genes preferentially targeted by RsAgo. We plan to further determine the role of nuclease in DNA processing in bacterial cells and test its interactions with RsAgo in vitro and in vivo.

Item Type:Article
Related URLs:
URLURL TypeDescription
Lisitskaya, L.0000-0002-5283-7983
Esyunina, D.0000-0002-3706-4425
Aravin, A.0000-0002-6956-8257
Kulbachinskiy, A.0000-0002-2292-6424
Additional Information:© 2021 The Authors. FEBS Open Bio © 2021 FEBS. Issue Online: 02 July 2021; Version of Record online: 02 July 2021. This work was supported in part by the Russian Science Foundation (grant 191400359).
Funding AgencyGrant Number
Russian Science Foundation191400359
Issue or Number:S1
Record Number:CaltechAUTHORS:20210821-161228936
Persistent URL:
Official Citation:(2021), Posters. FEBS Open Bio, 11: 103-507.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:110361
Deposited By: Tony Diaz
Deposited On:21 Aug 2021 16:25
Last Modified:16 Nov 2021 19:40

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