title: Mechanistic Insights into Archaeal and Human Argonaute Substrate Binding and Cleavage Properties.
abstract: Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation.
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Lübeck, 23538, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Willkomm
authors.affiliation: Department of Biochemistry, Genetics and Microbiology, Institute of Microbiology- Archaea Centre, University of Regensburg, Regensburg, 93053, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Zander
authors.affiliation: Department of Biochemistry, Genetics and Microbiology, Institute of Microbiology- Archaea Centre, University of Regensburg, Regensburg, 93053, Germany.
authors.forename: Dina
authors.initials: D
authors.lastname: Grohmann
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Lübeck, 23538, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage; D006801:Humans; D007700:Kinetics; D063747:Methanocaldococcus / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D060565:Argonaute Proteins; D004247:DNA; D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems