Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis.
The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.
Koike
M
M
Genome Research Group, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan. m_koike@nirs.go.jp
Awaji
T
T
Kataoka
M
M
Tsujimoto
G
G
Kartasova
T
T
Koike
A
A
Shiomi
T
T
D034961:Antigens, Nuclear; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000096:biosynthesis; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002877:Chromosomes, Human / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D000072200:Ku Autoantigen; D008677:Metaphase; D018613:Microscopy, Confocal; D008938:Mitosis; D009687:Nuclear Proteins / Q000032:analysis* / Q000096:biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen