Plasmid targeting and destruction by the DdmDE bacterial defence system.

While eukaryotic Argonautes play a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation (1). Here, we elucidate the activation pathway of the DNA Defense Module DdmDE system, which rapidly eliminates small, multicopy plasmids from Vibrio cholerae Seventh Pandemic Strain (7PET) (2). Through a combination of cryo-EM, biochemistry and in vivo plasmid clearance assays, we demonstrate DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that DdmD transitions from an autoinhibited, dimeric protein to monomers upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgo utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.