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J. Biol. Chem., Vol. 277, Issue 14, 11756-11764, April 5, 2002

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Repair of Sequence-specific ¹²⁵I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts^*

Andrea Odersky, Irina V. Panyutin^§, Igor G. Panyutin^§, Christian Schunck^¶, Elke Feldmann, Wolfgang Goedecke, Ronald D. Neumann^§, Günter Obe, and Petra Pfeiffer

From the  Institut für Genetik FB9, Universität Essen, Universitätsstrasse 5, D-45117 Essen, Germany and the ^§ Department of Nuclear Medicine, Warren G. Magnussen Clinical Center, National Institutes of Health, Bethesda, Maryland 20854

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 Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the ¹²⁵I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin ¹²⁵I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), 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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