Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair.
Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.
Fukushima
T
T
CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Konoe Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Takata
M
M
Morrison
C
C
Araki
R
R
Fujimori
A
A
Abe
M
M
Tatsumi
K
K
Jasin
M
M
Dhar
P K
PK
Sonoda
E
E
Chiba
T
T
Takeda
S
S
D000818:Animals; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D002645:Chickens; D002842:Chromatids / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000502:physiology*; D019281:Dimerization; D004307:Dose-Response Relationship, Radiation; D005091:Exons; D016195:G2 Phase; D005838:Genotype; D000072200:Ku Autoantigen; D051379:Mice; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D016196:S Phase
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D015739:Nocodazole