Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo.
Mammalian cells have an activity of mutagenic repair for DNA double-strand breaks (DSBs), microhomology-mediated end joining (MMEJ), in which DNA ends are joined via microhomologous sequences flanking the breakpoint. MMEJ has been indicated to be undertaken without Ku proteins, which are essential factors for non-homologous end joining (NHEJ). On the other hand, recent studies with cell-free (in vitro) systems indicated the involvement of Ku proteins in MMEJ, suggesting that MMEJ could be also undertaken by a Ku-dependent pathway. To clarify whether Ku proteins are essential in MMEJ in vivo, linearized plasmid DNAs with microhomologous sequences of 10bp at both ends were introduced as repair substrates into Ku80-proficient and Ku80-deficient CHO cells, and were subjected to MMEJ and NHEJ. Activities of MMEJ and NHEJ, respectively, of the cells were evaluated by mathematical modeling for the increase in fluorescence of GFP proteins produced from repaired products. The Ku80 deficiency caused approximately 75% reduction of the MMEJ activity in CHO cells, while it caused is > or =90% reduction of the NHEJ activity. Therefore, it was indicated that there is a Ku-dependent pathway for MMEJ; however, MMEJ is less dependent on Ku80 protein than NHEJ. The fraction of MMEJ products increased in proportion to the increase in the amounts of substrates. The results suggest that the increase in DSBs makes the cell more predominant for MMEJ. MMEJ might function as a salvage pathway for DSBs that cannot be repaired by NHEJ.
Katsura
Yukitaka
Y
Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
Sasaki
Shigeru
S
Sato
Masanori
M
Yamaoka
Kiyoshi
K
Suzukawa
Kazumi
K
Nagasawa
Toshiro
T
Yokota
Jun
J
Kohno
Takashi
T
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen