Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and non-homologous 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

BRCC36A is epistatic to BRCA1 in DNA crosslink repair and homologous recombination in Arabidopsis thaliana

BRCA1 is a well-known tumor suppressor protein in mammals, involved in multiple cellular processes such as DNA repair, chromosome segregation and chromatin remodeling. Interestingly, homologs of BRCA1 and several of its complex partners are also found in plants. As the respective mutants are viable, in contrast to mammalian mutants, detailed analyses of their biological role is possible. Here we demonstrate that the model plant Arabidopsis thaliana harbors two homologs of the mammalian BRCA1 interaction partner BRCC36, AtBRCC36A and AtBRCC36B. Mutants of both genes as well as the double mutants are fully fertile and show no defects in development. We were able to show that mutation of one of the homologs, AtBRCC36A, leads to a severe defect in intra- and interchromosomal homologous recombination (HR). A HR defect is also apparent in Atbrca1 mutants. As the Atbrcc36a/Atbrca1 double mutant behaves like the single mutants of AtBRCA1 and AtBRCC36A both proteins seem to be involved in a common pathway in the regulation of HR. AtBRCC36 is also epistatic to AtBRCA1 in DNA crosslink repair. Upon genotoxic stress, AtBRCC36A is transferred into the nucleus

Analyzing Somatic DNA Repair in Arabidopsis Meiotic Mutants

Meiotic and somatic recombination share a common set of factors. Thus, the analysis of somatic DNA repair in meiotic mutant lines should be of special interest. Growth defects of mutant plants induced by specific genotoxins can thereby hint to DNA repair functions of the affected proteins. Here, we describe two kinds of approaches to characterize deficiencies in DNA repair in mutant lines of Arabidopsis thaliana, after genotoxin treatment