Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells

Shigeru Sasaki, Masanori Sato, Yukitaka Katsura, Akihiro Kurimasa, David J. Chen, Shunichi Takeda, Hiroyuki Kuwano, Jun Yokota, Takashi Kohno

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)583-590
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume339
Issue number2
DOIs
StatePublished - Jan 13 2006

Keywords

  • DNA double-strand break
  • DT40
  • Homologous recombinational repair
  • Non-homologous end joining
  • Single-strand annealing

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Sasaki, S., Sato, M., Katsura, Y., Kurimasa, A., Chen, D. J., Takeda, S., Kuwano, H., Yokota, J., & Kohno, T. (2006). Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells. Biochemical and Biophysical Research Communications, 339(2), 583-590. https://doi.org/10.1016/j.bbrc.2005.11.052