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Originally published In Press as doi:10.1074/jbc.M111304200 on January 30, 2002
J. Biol. Chem., Vol. 277, Issue 14, 11756-11764, April 5, 2002
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.
*
This work was supported by Grant 96.053.2 from the
Wilhelm-Sander-Stiftung für Krebsforschung (to P. P.) and by
a fellowship of the Heisenberg-program of the Deutsche
Forschungsgemeinschaft (to P. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Present address: MetaSystems GmbH, Robert-Bosch-Str. 6, D-68804 Altlussheim, Germany.
To whom correspondence should be addressed: Institut für
Genetik FB9 (S05 T04 B26), Universität Essen,
Universitätsstr. 5, D-45117 Essen, Germany. E-mail:
petra.pfeiffer@uni-essen.de.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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