TY - JOUR
T1 - Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells
AU - Sasaki, Shigeru
AU - Sato, Masanori
AU - Katsura, Yukitaka
AU - Kurimasa, Akihiro
AU - Chen, David J.
AU - Takeda, Shunichi
AU - Kuwano, Hiroyuki
AU - Yokota, Jun
AU - Kohno, Takashi
N1 - Funding Information:
This study was partly supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare of Japan for the Third Term Comprehensive Control Research for Cancer, for Cancer Research (16S-1), and for Research on Human Genome and Tissue Engineering. M.S. and Y.K. were awardees of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan. We thank Dr. T. Matsuda of the Hall of Global Environmental Research, Graduate School of Global Environmental Studies, Kyoto University, Japan, for providing us with the pMY189 plasmid. We also thank the following scientists for providing us with cell lines: Drs. J.D. Minna, A.F. Gazdar, and M. Perucho. Cell lines were also obtained from the American Type Culture Collection. We also thank Ms. Eiko Sameshima for technical assistance.
PY - 2006/1/13
Y1 - 2006/1/13
N2 - A linearized plasmid DNA, in which tandem repeats of 400 bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.
AB - A linearized plasmid DNA, in which tandem repeats of 400 bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.
KW - DNA double-strand break
KW - DT40
KW - Homologous recombinational repair
KW - Non-homologous end joining
KW - Single-strand annealing
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U2 - 10.1016/j.bbrc.2005.11.052
DO - 10.1016/j.bbrc.2005.11.052
M3 - Article
C2 - 16310168
AN - SCOPUS:28444433111
SN - 0006-291X
VL - 339
SP - 583
EP - 590
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -