A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells.
DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.
Marangoni
E
E
Unité Propre de l'Enseignement Supérieur 'Radiosensibilité-Radiocarcinogenèse Humaine' (UPRES EA no. 2710, Pr. Eschwege), IFR no. 54, Institut Gustave Roussy, Villejuif, France.
Foray
N
N
O'Driscoll
M
M
Douc-Rasy
S
S
Bernier
J
J
Bourhis
J
J
Jeggo
P
P
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D002455:Cell Division / Q000235:genetics / Q000528:radiation effects*; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D006224:Cricetinae; D004265:DNA Helicases; D004260:DNA Repair; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D015870:Gene Expression; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D034961:Antigens, Nuclear; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen