Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state. [Display omitted] • SMC-based Wadjet defense systems directly survey DNA circularity by loop extrusion • Cryo-EM structures reveal a Wadjet cleavage state with distinct SMC motor geometry • Mechanical DNA bending by JetABC facilitates DNA kinking and cleavage by JetD • A single large DNA roadblock stalls SMC DNA translocation but not cleavage The JET nuclease (Wadjet) is a prokaryotic SMC-based defense system that exclusively cleaves plasmid DNA. Roisné-Hamelin et al. determine structures of plasmid-borne JET, elucidating DNA bending/kinking prior to cleavage. Together with biochemical work, this supports a model of plasmid recognition by SMC motor stalling when circular DNA is completely extruded. [ABSTRACT FROM AUTHOR]