biostudies-literatureHuang YNational Natural Science FoundationNatural Science Foundation of ChongqingChongqing Public Health Key Specialty (Discipline) Construction Projectgkag066https://www.ebi.ac.uk/biostudies/studies/S-EPMC12848934biostudies-literatureUnknown54(3)In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cosNucleic acids researchMycobacterial non-homologous end joining is required for antiphage defense.PMC128489348247232582072246CSTB2024NSCQ-MSX0703Liao YZhang THuang YHu JHuang ZHu HFan LXie JLi PXu HfalseMycobacterial non-homologous end joining is required for antiphage defense.In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos2026-01-01T00:00:00Z2026 Jan2026-06-14T03:25:35.858Z2026-06-14T03:15:37.097ZS-EPMC128489344160372910.1093/nar/gkag066