Molecular basis of the phosphorothioation-sensing antiphage defense system IscS-DndBCDE-DndI

Phosphorothioation serves as a DNA backbone modification mechanism, wherein a sulfur atom substitutes the nonbridging oxygen atom within the phosphodiester, facilitated by the gene products of dndABCDE or sspABCD. The combination of dndABCDE with dndFGH forms a bona fide defense system, where the DndFGH protein complex exhibits DNA nickase and DNA translocase activities to prevent phage invasion. In this study, we identified that dndI, co-transcribed with dndFGH, can independently couple with iscS-dndBCDE as an anti-phage defense system. Moreover, we resolved the crystal structure of DndI from Salmonella at a resolution of 3.10 Å. We discovered that its residue Y25, residing within a hydrophobic region of DndI, is involved in phosphorothioate (PT) sensing. Upon sensing PT modifications at 5'-GPSAAC-3'/5'-GPSTTC-3', the ATPase activity of DndI is stimulated, which subsequently triggers a conformational transition, facilitating the dissociation of DndI from self-DNA, thereby allowing DndI to avoid cleaving self-DNA while restricting PT-deficient phage DNA. This research broadens the knowledge of the mechanistic diversity underlying PT-based defense systems and highlights their complexity in the course of evolution.