Mechanistic Insights into Archaeal and Human Argonaute Substrate Binding and Cleavage Properties

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 n...

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Bibliographic Details
Published in:PloS one 2016-10, Vol.11 (10), p.e0164695-e0164695
Main Authors: Willkomm, Sarah, Zander, Adrian, Grohmann, Dina, Restle, Tobias
Format: Article
Language:English
Subjects:
Acids
Analysis
Archaea
Argonaute Proteins - chemistry
Argonaute Proteins - metabolism
Binding
Biochemistry
Biology and life sciences
Cleavage
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA Cleavage
Enzymes
Genes
Genetics
Homology
Humans
Kinases
Kinetics
Methanocaldococcus - genetics
Methanocaldococcus - metabolism
Methanocaldococcus jannaschii
Nucleic acids
Oligonucleotides - chemistry
Oligonucleotides - metabolism
Physical Sciences
Protein Binding
Proteins
Research and Analysis Methods
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems
Strands
Studies
Substrate Specificity
Substrates
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0164695