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

Biochem Biophys Res Commun. 2020 Dec 17;533(4):1484-1489. doi: 10.1016/j.bbrc.2020.10.051. Epub 2020 Oct 24.

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.

Keywords: Argonaute proteins; DNA targeting; Small DNA; Small RNA; pAgo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Argonaute Proteins / chemistry
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • Protein Binding
  • RNA, Guide, CRISPR-Cas Systems
  • Rhodobacter sphaeroides / genetics
  • Rhodobacter sphaeroides / metabolism*

Substances

  • Argonaute Proteins
  • Bacterial Proteins
  • DNA