No abstract

No abstract

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.

Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer‐killing effects. Accumulating evidence shows that DNA double‐strand break ( DSB ) repair proteins, which function in Ku‐dependent non‐homologous DNA ‐end joining ( NHEJ ), are potential target molecules for next‐generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku‐dependent NHEJ . Here, we cloned and sequenced feline XLF cDNA and characterized X‐ray repair cross‐complementing protein 4‐like factor ( XLF ), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser‐induced DSB sites in a Ku‐dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14‐3‐3‐binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF . Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku‐dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells, and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 and Ku80, might play a crucial role in regulating NHEJ activity. Recently, companion animals, such as canines, have been proposed to be a good model in many aspects of cancer research. However, the localization and regulation mechanisms of core NHEJ factors in canine cells have not been elucidated. Here, we show that the expression and subcellular localization of canine XRCC4 changes dynamically during the cell cycle. Furthermore, EYFP-canine XRCC4 accumulates quickly at laser-microirradiated DSB sites. The structure of a putative human XRCC4 nuclear localization signal (NLS) is highly conserved in canine, chimpanzee and mouse XRCC4. However, the amino acid residue corresponding to the human XRCC4 K210, thought to be important for nuclear localization, is not conserved in canine XRCC4. Our findings might be useful for the study of the molecular mechanisms of Ku-dependent NHEJ in canine cells and the development of new radiosensitizers that target XRCC4.