DNA-guided DNA interference by a prokaryotic Argonaute

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• RNA interference (RNAi) is a conserved eukaryotic process utilizing small RNA molecules to guide Argonaute (Ago) proteins for gene regulation and defense.
• The function of prokaryotic Ago homologs, despite predicted roles in host defense, remains largely uncharacterized.
• Bioinformatic analyses suggested potential involvement of prokaryotic Ago proteins in host defense mechanisms.

Purpose of the Study:

• To investigate the functional role of the Argonaute protein from the bacterium Thermus thermophilus (TtAgo).
• To elucidate the mechanism by which TtAgo mediates host defense in prokaryotes.
• To determine if TtAgo utilizes RNA or DNA guides for target recognition and cleavage.

Main Methods:

• In vivo characterization of TtAgo activity in Thermus thermophilus.
• Analysis of small nucleic acid guides associated with TtAgo.
• Biochemical assays to determine DNA target cleavage by TtAgo-guide complexes.

Main Results:

• TtAgo is loaded with 5'-phosphorylated DNA guides, 13-25 nucleotides in length.
• These DNA guides exhibit a strong bias for a 5'-end deoxycytidine and are often plasmid-derived.
• TtAgo, guided by these small DNAs, effectively cleaves complementary DNA strands, demonstrating DNA-guided DNA interference.

Conclusions:

• The Argonaute protein from Thermus thermophilus (TtAgo) functions as a DNA-guided DNA interference system.
• TtAgo acts as a barrier against the uptake and propagation of foreign DNA in prokaryotes.
• Despite structural similarities to eukaryotic RNA interference machinery, TtAgo employs DNA guides for DNA target cleavage, highlighting a distinct prokaryotic defense strategy.