OLD sentinel: an abortive tRNase surveys phage replication and DNA defects in RecBCD-compromised cells

Abstract

OLD, an abortive immunity protein from prophage P2, consists of an ABC ATPase sensor and a TOPRIM nuclease effector -- a core architecture shared by a large protein family, including components of antiphage systems Gabija, PARIS, Septu, and Lamassu. OLD was originally identified for its lethality in recB-deficient cells and inhibition of bacteriophage {lambda} infection, but the mechanisms governing its activation have remained elusive. Here, we present the cryo-EM structure of an inactive OLD tetramer and show that destabilization into dimeric form opens the TOPRIM catalytic site, stimulating tRNA cleavage. This activity arrests translation, a phenotype rescued by phage-encoded tRNAs. We demonstrate that OLD activation is not strictly RecBCD-dependent: OLD binds aberrant DNA structures in recB-deficient cells, but activation during infection requires recognition of single-stranded DNA hairpins at the phage replication origin. Collectively, our findings reveal how host and phage DNA processing factors create a complex landscape controlling OLD-mediated immunity.