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

Autor: Odersky, Andrea, Panyutin, Irina V., Panyutin, Igor G., Schunck, Christian, Feldmann, Elke, Goedecke, Wolfgang, Neumann, Ronald D., Obe, Günter, Pfeiffer, Petra
Zdroj: Journal of Biological Chemistry; April 2002, Vol. 277 Issue: 14 p11756-11764, 9p
Abstrakt: 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 the125I-TFO within its target site. We show that NHEJ of125I-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 vivoafter125I-TFO-mediated radiodamage indicates that our in vitrosystem may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.
Databáze: Supplemental Index