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doi:10.1534/genetics.106.065185
A more recent version of this article appeared on January 1, 2007.
REGULAR RESEARCH PAPERS |
Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and non-homologous end-joining
Michael Pacher 1, Waltraud Schmidt-Puchta 1 and Holger Puchta 1*
1 Universität Karlsruhe
* To whom correspondence should be addressed. E-mail: holger.puchta{at}bio.uka.de.
Submitted on August 31, 2006
Revised on September 28, 2006
Accepted on 6 October 2006
Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single DSB in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes each carrying an I-SceI site. After transient expression of I-SceI a kanamycin resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by non-homologous end-joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite of the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.
Key Words: DNA repair, I-SceI, Translocation, gene technology, single-strand annealing
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