Abstract

Bacteria have evolved a myriad of host-defense systems that protect against invasive mobile genetic elements. The DdmDE module, which consists of a DNA-guided, DNA-targeting prokaryotic Argonaute (pAgo) DdmE and a helicase-nuclease enzyme DdmD, represents a model anti-plasmid system. Here, we demonstrate that a double-stranded DNA (dsDNA)-destabilizing force generates transient bubbles that provoke promiscuous binding by DNA-guided DdmE. The resulting nucleoprotein complex discriminates on-targets from off-targets primarily through differences in dissociation rates. Remarkably, DNA-bound DdmE recruits a DdmD dimer to drive dsDNA shortening against a resisting force. Further analysis attributes this shortening to target-centered, bidirectional dsDNA unwinding and single-stranded DNA (ssDNA) extrusion by DdmD, in which monomeric DdmD engages both unwound strands. During this process, free DdmD rapidly associates with the two extruded strands and catalyzes ssDNA digestion with a sequence preference for a 5′ guanine. Our findings provide a dynamic perspective on how pAgos cooperate with accessory factors to achieve plasmid clearance.