Two Unlinked Double-Strand Breaks Can Induce Reciprocal Exchanges in Plant Genomes via Homologous Recombination and Nonhomologous End Joining

Abstract

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.

In planta gene targeting

“…Southern blotting of EcoRI-, HindIII-, or MfeI-digested genomic DNA (F3′ generation) using the membrane "Hybond N+" (GE Healthcare) was performed as described (24). The DNA probes were labeled as described (41). Probe A was PCR-amplified from VC-SBT359-6qcz using the oligonucleotides 5′-GTTGTGGGAGGTGATGTC-3′ and 5′-CCGAGAACGTCATCACCG-3′; probe B from VC-SBT366-12qcz using the oligonucleotides 5′-CTTGGATTGAACAAGATGGATTGC-3′ and 5′-CAGA-AGAACTCGTCAAGAAGGCG-3′.…”

Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

“…Thus, our results indicate that within the GU.US construct, most DSBs are repaired by an interchromatid process using the sister helix as a template and are linked with reciprocal exchange. In contrast to the presence of two or more DSBs per nucleus, a single DSB per nucleus is not assumed to significantly increase the frequency of reciprocal chromosome translocations (Puchta, 1999;Richardson and Jasin, 2000;Pacher et al, 2007). We observed chromatin bridges in 1.6% of 126 anaphases in GU.US control meristems and 3.8% of 105 anaphases of the I-SceI expressing plants (not significant at a 99% confidence interval).…”

The Origin of a “Zebra” Chromosome in Wheat Suggests Nonhomologous Recombination as a Novel Mechanism for New Chromosome Evolution and Step Changes in Chromosome Number

“…The alloplasmic wheat plant was double monosomic for 5A and 1H t S and the two nonhomologous chromosomes may have prealigned prior to S-phase of meiosis or mitosis in germinal tissues during transition from the vegetative to reproductive phase. Chromosome z5A might have originated from nonhomologous recombination during the DNA double-strand break (DSB) repair process (Gorbunova and Levy 1999;Pacher et al 2007) during the S-phase. Initially, at least five breaks occurred in chromosome 5A, resulting in six chromatin blocks.…”