TY - JOUR
T1 - Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay
AU - Secretan, M. B.
AU - Scuric, Z.
AU - Oshima, J.
AU - Bishop, A. J.R.
AU - Howlett, N. G.
AU - Yau, D.
AU - Schiestl, R. H.
N1 - Funding Information:
For excellent technical assistants for FACS analysis we would like to thank Amy Imrich at FACS Core Facility at Harvard School of Public Health and Iris Williams at FACS Core Facility at UCLA School of Medicine. We thank M.Z. Zdzienicka for kindly supplying cell lines. This work was supported by grants from the National Institute of Environmental Health Sciences, NIH, RO1 Grant No. ES09519 and KO2 award ES00299, both to RHS. M.B.S. was a recipient of a Post-doctoral fellowship from the Swiss Cancer League and A.J.R.B was a recipient of NIH RCDA Award No. F32GM19147.
PY - 2004/10/4
Y1 - 2004/10/4
N2 - In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.
AB - In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.
KW - DNA double-strand breaks
KW - DSB
KW - EGFP
KW - EYFP
KW - HR
KW - MCS
KW - NHEJ
KW - PSS
KW - double-strand break(s)
KW - enhanced green fluorescent protein
KW - enhanced yellow fluorescent protein
KW - homologous recombination
KW - multiple cloning site
KW - non-homologous end-joining
KW - protruding single strands
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U2 - 10.1016/j.mrfmmm.2004.05.016
DO - 10.1016/j.mrfmmm.2004.05.016
M3 - Article
C2 - 15450431
AN - SCOPUS:4344660051
SN - 0027-5107
VL - 554
SP - 351
EP - 364
JO - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
IS - 1-2
ER -