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Ku, a heterodimer of Ku70 and Ku80, plays a key role in multiple nuclear processes, e.g. DNA repair, chromosome maintenance, and transcription regulation. Heterodimerization is essential for Ku-dependent DNA repair in vivo, although its role is poorly understood. Some lines of evidence suggest that heterodimerization is required for the stabilization of Ku70 and Ku80. Here we show that the heterodimerization of these Ku subunits is important for their nuclear entry. When transfected into Ku-deficient xrs-6 cells, exogenous Ku70 and Ku80 tagged with green fluorescent protein accumulated into the nucleus, whereas each nuclear localization signal (NLS)-dysfunctional mutant was undetectable in the nucleus, supporting the idea that each Ku can translocate to the nucleus through its own NLS. On the other hand, the nuclear accumulation of each NLS-dysfunctional mutant was markedly enhanced by the presence of an exogenous wild-type counterpart. In Ku-expressing HeLa cells, each NLS-dysfunctional mutant, as well as wild-type Ku70 and Ku80, was still detectable in the nucleus, whereas the double mutant of each Ku subunit with decreased functions of both nuclear targeting and dimerization was undetectable in the nucleus. Our results indicate that each Ku subunit can translocate to the nucleus not only through its own NLS but also through heterodimerization with each other.

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The skin is an external organ that is most frequently exposed to radiation. High-dose radiation initiates and promotes skin cancer and acute radiation injury. It is important to investigate the influence of high-dose radiation exposure on the skin at the molecular level to understand acute radiation injury. To identify genes that are associated with injury caused by high-dose radiation exposure of the skin, we used microarray technology to examine the effect of irradiation on approximately 1000 genes in normal human epidermal keratinocytes at 3 h postirradiation with a cytotoxic dose of X-ray (5 Gy). We found that 16 and 59 genes were up- and down-regulated respectively in the keratinocytes. Several apoptosis-related genes, for example, BAK and TSC-22, and anti-proliferative genes, for example, BTG-1 and BTG-3, were up-regulated. We focused on ATF3 because ATF3 is induced most strongly by X-irradiation, and its function in keratinocytes is unknown. The induction of the ATF3 mRNA and protein in keratinocytes following X-ray was confirmed by RT-PCR and western blot analysis. ATF3 was also induced and accumulated within the nuclei of keratinocytes after X-ray irradiation in vivo and in vitro. Exogenous EYFP-ATF3 also accumulated within the nuclei of keratinocytes. In the transient expression assay, EYFP-ATF3, but not EYFP, induced apoptosis in keratinocytes. Taken together, these results suggest that ATF3 plays a role in apoptosis in keratinocytes and is associated with skin injury caused by ionizing radiation.

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.

The Ku protein is a complex of two subunits, Ku70 and Ku80, and it plays a role in multiple nuclear processes, e.g., nonhomologous DNA-end-joining (NHEJ), chromosome maintenance, and transcription regulation. On the other hand, several studies have reported a cytoplasmic or cell surface localization of Ku in various cell types. The mechanism underlying the regulation of all the diverse functions of Ku is still unclear, though the mechanism that regulates the nuclear localization of Ku70 and Ku80 appears to play, at least in part, a key role in regulating the physiological function of Ku. In this study, we generated cell lines expressing the human Ku80 tagged with the green fluorescent protein (GFP) color variants in Ku80-deficient cells, i.e., xrs-6 derived from CHO-K1. Although Ku70, as well as Ku80, was undetectable in xrs-6 cells, it was seen in these transformants at a level similar to the level of CHO-K1. Furthermore, etoposide- and radiosensitive phenotype of xrs-6 cells were corrected by an introduction of the tagged Ku80. Moreover, the tagged Ku80 suppressed apoptosis triggered by DNA damage. These results demonstrate that fusion to the GFP color variants does not interfere with the functions of the Ku80 in the Ku-dependent DSB repair. Therefore, these transformants might be useful not only in the analysis of Ku80 behavior, but also in an analysis of the dynamics of the NHEJ repair process.

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