Molecularly targeted therapies have high specificity and significant cancer‐killing effect. However, their antitumor effect might be greatly diminished by variation in even a single amino acid in the target site, as it occurs, for example, as a consequence of SNPs. Increasing evidence suggests that the DNA repair protein Ku80 is an attractive target molecule for the development of next‐generation radiosensitizers for human cancers. However, the localization, post‐translational modifications (PTMs), and complex formation of Ku80 have not been elucidated in canines. In this study, for the first time, we cloned, sequenced, and characterized canine Ku80 cDNA. Our data show that canine Ku80 localizes in the nuclei of interphase cells and is quickly recruited at laser‐induced double‐strand break sites. Comparative analysis shows that canine Ku80 had only 82.3% amino acid identity with the homologous human protein, while the nuclear localization signal (NLS) in human and canine Ku80 is evolutionarily conserved. Notably, some predicted PTM sites, including one acetylation site and one sumoylation site within the NLS, are conserved in the two species. These findings suggest that the spatial and temporal regulation of Ku80 might be conserved in humans and canines. However, our data indicate that the expression of Ku80 is considerably lower in the canine cell lines examined than in human cell lines. These important findings might be useful to better understand the mechanism of the Ku80‐dependent DNA repair and for the development of potential next‐generation radiosensitizers targeting common targets in human and canine cancers.
