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Papers In Press, published online ahead of print January 30, 2002
J. Biol. Chem, 10.1074/jbc.M111304200
Submitted on November 27, 2001
Revised on January 30, 2002
Accepted on January 28, 2002
Institute of Genetics FB9 (S05 T04 B26), University of Essen, Essen D-45117
Corresponding Author: petra.pfeiffer{at}uni-essen.de
In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of 125I positioned against a specific target-site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from CHO cells. The efficiency and quality of NHEJ of the “complex” DSB induced by the 125I-TFO was compared to that of “simple” DSB induced by restriction enzymes (RE). We demonstrate that the extracts are indeed able to rejoin 125I-TFO-induced DSB although at approximately ten-fold decreased efficiency compared to RE-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the 125I-TFO within its target-site. We show that NHEJ of 125I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after 125I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.
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