Impact of amino acid substitutions in two functional domains of Ku80: DNA-damage-sensing ability of Ku80 and survival after irradiation

Abstract

Various chemotherapeutic drugs, such as etoposide, and ionizing radiation (IR) have been clinically applied for the treatment of many types of animal and human malignancies. IR and chemotheraputic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). On the other hand, unrepaired or incorrectly repaired DSBs can lead to chromosomal truncations and translocations, which can contribute to the development of cancer in humans and animals. Thus, it is important to clarify the molecular mechanisms underlying the chemosensitivity or radiosensitivity of mammalian cells in order to develop medical treatments and next-generation chemotherapeutic drugs for cancer. Previously, we established and analyzed cell lines stably expressing chimeric constructs of EGFP and the wild-type Ku80 (XRCC5) protein or its mutant protein to which mutations were introduced by the site-directed mutagenesis. We found that the Ku70 (XRCC6)-binding-site mutations (A453H/V454H) of Ku80 and nuclear localization signal (NLS)-dysfunctional mutations (K565A/ K566A/K568A) affected the ability to complement etoposide sensitivity. In this study, we examined the radiosensitivity of these cell lines. We found that either or both amino acid substitutions in two functional domains of Ku80, i.e., Ku70-binding-site mutations (A453H/ V454H) and NLS-dysfunctional mutations (K565A/K566A/K568A), affect the ability to complement radiosensitivity. Moreover, these mutations in the two domains of Ku80 affect the DSB-sensing ability of Ku80. These information and Ku80 mutant cell lines used might be useful for the study of not only the dynamics and function of Ku80, but also the molecular mechanism underlying the cellular response to IR and chemotherapeutic drugs in mammalian cells. © 2014 The Japanese Society of Veterinary Science.