[3S02a] 05. Genome Editing Technology and its Applications - Part 2

2:45 PM - 3:10 PM JST(5:45 AM - 6:10 AM UTC)

[3GET-10] The Microbial PIWI-RE System: A Multi-Component Complex Defends against Plasmids and Promotes Homologous Recombination

○Yan Feng¹ (1. Shanghai Jiao Tong University (China))

Keywords:

Bacteria defense,Argonaute,PIWI family,Multi-enzyme complex,Homologous recombination

[Purpose] Bacteria have evolved diverse innate defense systems to maintain genomic integrity against aggression of invasive mobile genetic elements (MGEs) such as plasmids and phages. Prokaryotic argonautes (pAgos), belonging to PIWI family, is well recognized as a typical guide−dependent defense module to cleave the single DNA chain. However, there is still a mystery how they act on the double strands MGEs. We noticed that the PIWI-RE clade, exhibiting a distinct genetic cluster with a restriction endonuclease (REase) and an SFII−family helicase (DinG), remains unknown. Here, we characterize the REase, DinG and PIWI−RE complexes (RDP) from an environmentally ubiquitous bacterium and an opportunistic human pathogen to reveal the novel defense mechanism and further explore their potential for biotechnological applications.
[Methods] Phylogenetic analysis; enzyme assays; high−throughput sequencing; size-exclusion chromatography; multiangle light scattering; plasmid interference assays; homologous recombination assays.
[Results] We first conducted phylogenetic analysis for an independent PIWI−RE evolutionary clade of pAgos. Notibly, a close co −evolutionary relationship exists among the REase, DinG, and PIWI−RE proteins. Following the overexpression and purification of evolutionarily representative RDP homologs, we found that both of them assemble into stable hexameric complexes. The complexes exhibited guide−dependent ssDNA and dsDNA cleavage activities, significantly enhancing nuclease activity compared to its individual components. Furthermore, we revealed that the RDP system exhibits broad defense capabilities against plasmids with different copy numbers and phages. We proposed that the RDP system mediated a coordinated “recognition (PIWI-RE) −unwinding (DinG) − cleavage (REase)” process, which differs from the conventional pAgo defense systems. By co−expressing with lambda-red recombinase and single−stranded DNA−binding protein in E. coli, RDP system could significantly promote homologous recombination efficiency.
[Consideration] The PIWI−RE clade represents a novel multi−component defense system whose molecular mechanisms-including precise DNA recognition, recruitment of DinG and REase, and allosteric communication within the hexameric complex−remain to be fully elucidated. Cryo−electron microscopy and single−molecule analyses are currently underway.
[Conclusion] Our findings identify the RDP system as a novel, multi−component prokaryotic defense machinery that expands pAgo−mediated immunity. The system provides broad−spectrum defense against mobile genetic elements and enhances homologous recombination, offering both biological insight and biotechnological utility.