Antiviral defence is a conserved function of diverse bacterial DNA glycosylases.

Bacteria are frequently attacked by viruses, known as phages, and rely on diverse defence systems to survive. While phages can evade defences by covalently modifying their DNA, these non-canonical nucleobases create molecular signatures that bacteria can exploit. Here, using structure-guided discovery, we identified two widespread families of anti-phage DNA glycosylases, Dag1 and Dag2. Although DNA glycosylases are classically associated with DNA repair, Dag1 and Dag2 act as antiviral effectors that selectively target phages carrying modified guanine bases. Guided by the conserved glycosylase fold, we uncovered numerous defence-associated glycosylases that collectively form a diverse repertoire of enzymes targeting chemically modified phage DNA. We further identified a distinct glycosylase superfamily that protects against phages carrying modified thymidine bases. Together, these findings establish DNA glycosylases as a versatile class of bacterial immune proteins and highlight structure-guided discovery as a powerful strategy for uncovering hidden antiviral defences.