Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining.

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (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 nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite 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.

Intrachromosomal homologous recombination in Arabidopsis thaliana.

Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an important task for plant biology, is becoming feasible. An assay system based on transgenes harboring homologous overlaps of the beta-glucuronidase (uidA) gene is available to determine recombination behavior in various mutant backgrounds. Restoration of the marker gene by homologous recombination can be detected by histochemical staining in planta. Inclusion of a site of the rare cutting restriction enzyme I-SceI in the transgene construct enables the determination of recombination frequencies after induction of double-strand breaks. In this chapter we describe how the respective transgene is transferred by transformation or crossing into the mutant background, how recombination frequencies are determined, and, if necessary, how cells carrying a restored uidA gene can be isolated and propagated for molecular analysis of the particular recombination event.