The establishment and characterization of cell lines stably expressing human Ku80 tagged with enhanced green fluorescent protein.
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.
Koike
Manabu
M
Radiation Hazards Research Group, National Institute of Radiological Sciences, Chiba, Japan. m_koike@nirs.go.jp
Koike
Aki
A
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology* / Q000528:radiation effects*; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D049452:Green Fluorescent Proteins; D000072200:Ku Autoantigen; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011829:Radiation Dosage; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014018:Tissue Distribution
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen