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

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. ; © 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. ; Supplemental Material - 1-s2.0-S0006291X20319690-mmc1.pdf