Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells.
A linearized plasmid DNA, in which tandem repeats of 400bp 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.
Sasaki
Shigeru
S
Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
Sato
Masanori
M
Katsura
Yukitaka
Y
Kurimasa
Akihiro
A
Chen
David J
DJ
Takeda
Shunichi
S
Kuwano
Hiroyuki
H
Yokota
Jun
J
Kohno
Takashi
T
D001483:Base Sequence; D001681:Biological Assay; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D013997:Time Factors
D004247:DNA