KU80 suppresses endonuclease G activity to preserve genomic integrity

Endonuclease G (ENDOG) is involved in DNA replication, mitochondrial DNA maintenance, and late apoptosis. Its role in genome maintenance and cell death necessitates strict regulation to prevent unwanted DNA damage. Although inhibitors of ENDOG have been identified in nonmammalian species, no such inhibitors have been discovered in mammals. Here, we identify KU80 as a novel negative regulator of ENDOG in human cells. The KU80 C‐terminal domain (CTD) shares structural homology with Drosophila EndoG inhibitor (EGI), directly binds ENDOG and inhibits its nuclease activity in vitro . In silico modeling and immunoprecipitation reveal the KU80 core domain mediates ENDOG interaction. Depletion of KU80 increases nuclear ENDOG accumulation and DNA damage under normal conditions, evidenced by elevated phosphorylated histone H2AX (γH2AX) levels. Conversely, ENDOG‐deficient cells show reduced DNA damage and increased chemotherapeutic resistance. We uncover a mechanism where KU80 maintains genomic stability by inhibiting ENDOG activity and nuclear translocation. This study establishes KU80 as a key DNA damage response regulator, offering insights into potential therapies for diseases linked to dysregulated ENDOG activity.