Presentation Information
[P03-323]A Guide−Directed DNA Repair Machine Promotes Bacterial Genome Stability and Homologous Recombination
○Jiayu Wei1,2, Mengyu Li1, Hao Yang1, Yan Feng1 (1. School of Life Sciences and Biotechnology, Shanghai Jiao Tong University (China), 2. Hainan Research Institute, Shanghai Jiao Tong University (China))
Keywords:
Prokaryotic Argonaute,DNA Repair,Genome Stability,Homologous Recombination,Gene Editing
[Purpose] Bacterial genomes have constantly faced the dual threats of exogenous mobile genetic element (MGE) invasion and endogenous DNA damage throughout long evolutionary history. To counteract damage caused by metabolic byproducts and replication errors, bacteria have evolved intricate and sophisticated DNA repair networks to maintain genome integrity. Prokaryotic Argonaute (pAgo) systems play a pivotal role in this process, particularly the PIWI−RE branch. This branch, distinct from traditional immune systems in its genetic composition, comprises a PIWI−RE protein, a restriction endonuclease (REase), and a SFII family helicase (DinG), collectively forming the complex (RDP), which is highly conserved across diverse bacterial species. However, the molecular mechanisms and physiological functions of this branch remain largely unknown. Our study elucidates a novel DNA repair mechanism involving the RDP complex and further explores its potential applications in developing gene editing technologies.
[Method] Enzyme assays, high−throughput sequencing, multiangle light scattering, RNA−Seq, DNA repair assays, SOS response detection assay, flow cytometry analysis, gene editing assays.
[Results] Our research indicate that both individual genes and the complete operon of RDP significantly enhance bacterial survival and promote bacterial growth after DNA damage, with the complete operon exhibiting the strongest protective effect. The deletion of recA completely abolished this repair phenotype, while the disruption of recBCD or recFOR only led to a partial decrease in repair capability. Transcriptome analysis revealed that RDP significantly upregulates the expression of homologous recombination related genes. In vitro biochemical experiments confirmed that REase possesses nuclease activity, DinG exhibits 5′→3′ helicase function, and PIWI is a guide RNA−guided DNA−binding nuclease. Based on these results, we propose a stepwise repair model: damage recognition−translocation and unwinding−precise excision−homologous recombination, thereby maintaining endogenous genome stability. Furthermore, introducing Chi sequences into the target sequences can further enhance the recombination efficiency of RDP−mediated resistance genes and fluorescent reporter genes.
[Consideration] The RDP complex functions as a multi−component DNA damage repair system, yet the precise molecular mechanisms underlying its stepwise repair cascade and the coupling of the complex with the host DNA repair machinery to facilitate faithful repair−remain to be fully characterized. Dynamic single−molecule approaches are presently in progress.
[Conclusion] Our findings unveils an RDP molecular machine that orchestrates guide−directed DNA repair processing with host recombination machinery, providing new mechanistic insights into bacterial genome stability and establishing a potential platform for precision gene editing tools.
[Method] Enzyme assays, high−throughput sequencing, multiangle light scattering, RNA−Seq, DNA repair assays, SOS response detection assay, flow cytometry analysis, gene editing assays.
[Results] Our research indicate that both individual genes and the complete operon of RDP significantly enhance bacterial survival and promote bacterial growth after DNA damage, with the complete operon exhibiting the strongest protective effect. The deletion of recA completely abolished this repair phenotype, while the disruption of recBCD or recFOR only led to a partial decrease in repair capability. Transcriptome analysis revealed that RDP significantly upregulates the expression of homologous recombination related genes. In vitro biochemical experiments confirmed that REase possesses nuclease activity, DinG exhibits 5′→3′ helicase function, and PIWI is a guide RNA−guided DNA−binding nuclease. Based on these results, we propose a stepwise repair model: damage recognition−translocation and unwinding−precise excision−homologous recombination, thereby maintaining endogenous genome stability. Furthermore, introducing Chi sequences into the target sequences can further enhance the recombination efficiency of RDP−mediated resistance genes and fluorescent reporter genes.
[Consideration] The RDP complex functions as a multi−component DNA damage repair system, yet the precise molecular mechanisms underlying its stepwise repair cascade and the coupling of the complex with the host DNA repair machinery to facilitate faithful repair−remain to be fully characterized. Dynamic single−molecule approaches are presently in progress.
[Conclusion] Our findings unveils an RDP molecular machine that orchestrates guide−directed DNA repair processing with host recombination machinery, providing new mechanistic insights into bacterial genome stability and establishing a potential platform for precision gene editing tools.
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