RESUMO
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.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , DNA/química , DNA/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ligação Proteica , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Rhodobacter sphaeroides/genéticaRESUMO
Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.