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

• Published in: The Journal of biological chemistry 2002-04, Vol.277 (14), p.11756-11764
• Main Authors: Odersky, A, Panyutin, I V, Panyutin, I G, Schunck, C, Feldmann, E, Goedecke, W, Neumann, R D, Obe, G, Pfeiffer, P
• Format: Article
• Language: English
• ISSN: 0021-9258

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 super(125)I 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 super(125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin super(125)I-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 super(125)I-TFO within its target site. We show that NHEJ of super(125)I-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 super(125)I-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.