Structural and functional characterization of Staphylococcus aureus RecJ reveals CTD ‐mediated functional divergence in DNA repair mechanisms

Abstract Staphylococcus aureus is a major human pathogen that tolerates diverse environmental stresses, and multidrug‐resistant strains pose serious clinical challenges. RecJ, a nuclease involved in DNA repair, contributes to stress resistance, yet its structural and mechanistic features in S. aureus remain unclear. Here, we classified bacterial RecJ proteins into six subtypes based on the geometry of their C‐terminal domains (CTDs) and performed structural and biochemical characterization of S. aureus RecJ (SaRecJ, SA‐type) in comparison with Deinococcus radiodurans RecJ (DrRecJ, DR‐type). Biochemical assays revealed that SaRecJ is a Mg 2+ ‐dependent 5′–3′ exonuclease that remains active under high‐temperature and high‐salt conditions, consistent with the stress‐tolerant physiology of S. aureus . The 2.8 Å crystal structure of SaRecJ revealed distinctive CTD conformations and the absence of the canonical SSB‐binding pocket observed in DrRecJ. Functional analyses further suggested that the CTD contributes to SaRecJ stability and long ssDNA resection. Unlike DrRecJ, SaRecJ specifically participates in the repair of mitomycin C (MMC)—induced DNA cross‐links. Moreover, SaRecJ cooperates with the helicase—nuclease SaDinG for coordinated DNA end resection. Collectively, these results define a CTD‐driven functional specialization of RecJ and reveal how S. aureus remodels its DNA repair machinery to maintain genome integrity under cross‐linking stress.