1. Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein.
- Author
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Lisitskaya L, Petushkov I, Esyunina D, Aravin A, and Kulbachinskiy A
- Subjects
- Argonaute Proteins chemistry, Argonaute Proteins genetics, Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, DNA chemistry, DNA genetics, Models, Molecular, Nucleic Acid Conformation, Nucleic Acid Denaturation, Protein Binding, Rhodobacter sphaeroides genetics, RNA, Guide, CRISPR-Cas Systems, Argonaute Proteins metabolism, Bacterial Proteins metabolism, DNA metabolism, Rhodobacter sphaeroides metabolism
- 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., Competing Interests: Declaration of competing interest 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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