Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts.
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 (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 (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (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 (125)I-TFO within its target site. We show that NHEJ of (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 (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.
Odersky
Andrea
A
Institut für Genetik FB9, Universität Essen, Universitätsstrasse 5, D-45117 Essen, Germany.
Panyutin
Irina V
IV
Panyutin
Igor G
IG
Schunck
Christian
C
Feldmann
Elke
E
Goedecke
Wolfgang
W
Neumann
Ronald D
RD
Obe
Gunter
G
Pfeiffer
Petra
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014981:Xenopus
D004247:DNA