A novel DNA-protective function of Escherichia coli thioredoxin 2 mediated by its N-terminal zinc-binding domain.

Thioredoxins are ubiquitous thiol-disulfide oxidoreductases that maintain intracellular redox homeostasis. In addition to its conserved catalytic domain, Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain whose physiological function remains largely unknown. Here, we identify a previously unrecognized DNA-binding activity of EcTrx2 and demonstrate its role in protecting DNA during oxidative stress. Electrophoretic mobility shift assays showed that EcTrx2 bound plasmid DNA in a concentration-dependent and GST-tag-independent manner, whereas EcTrx1 exhibited no detectable DNA-binding activity. DNA binding was abolished by deletion of the N-terminal zinc-binding domain and was blocked by zinc occupancy, indicating that this unique domain is essential for DNA interaction. Consistent with these findings, EcTrx2 significantly protected plasmid DNA from DNase I digestion and hydroxyl radical-mediated oxidative damage in vitro. Furthermore, EcTrx2 enhanced bacterial tolerance to the DNA-damaging agents zeocin and diamide, supporting the physiological relevance of its DNA-binding activity. Our results reveal a DNA-binding role for EcTrx2 and identify its N-terminal zinc-binding domain as a key determinant of DNA binding and protection against oxidative DNA damage.