An asymmetric helicase dimer drives 3′-to-5′ DNA unwinding in type II Druantia antiphage defence
Abstract
Druantia systems are widespread antiphage defence systems organized around the conserved helicase DruE. However, how DruE recognizes DNA substrates and executes phage restriction has remained unclear, particularly for type II Druantia systems that encode DruE together with DruM, DruF, and DruG. Here we show that DruE from the type II Druantia system of Pseudomonas protegens is a DNA-stimulated ATPase and 3′-to-5′ helicase that preferentially engages substrates with an exposed 3′ single-stranded entry strand at a duplex junction, such as 3′-overhang and forked DNA. Seven cryo-EM structures, including an apo dimer and six DNA-bound nucleotide states, define a nucleotide-resolved structural trajectory for substrate loading, motor remodeling, displaced-strand capture, and DNA unwinding. DruE forms an asymmetric homodimer in which one protomer functions as the tracking-strand motor, whereas the second engages the displaced strand and adjacent duplex in a clamp-like configuration. A fork-junction anchor, a nucleotide-gated lid, an Arg1422-associated contact switch, and inter-protomer contacts together explain how ATP turnover is coupled to directional unwinding. Because DruE recognizes fork-like DNA structures that may also arise during host DNA replication or repair, its activity likely requires regulation. Consistent with this idea, mitomycin C (MMC) sensitivity phenotypes suggest that the type II accessory proteins DruM, DruF, and DruG may regulate DruE activity or limit self-toxicity. Together, these results define a mechanochemical model for type II DruE and support a model in which Druantia targets 3′-ended or fork-like phage replication intermediates to suppress infection.References
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