RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.

Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.Gene targeting (GT) involves the disruption or replacement of an endogenous allele by one manipulated in vitro. This replacement requires that after transfection into the target cell the transgene recombines with the target allele by homologous recombination (HR) by virtue of sharing extensive sequence similarity, rather than integrating randomly by illegitimate recombination. As HR occurs efficiently in many lower eukaryotes such as budding yeast and in bacteria, GT has proved to be a very powerful reverse genetic tool to study gene function. The GT technology is also now routine, if not still laborious in the generation of transgenic knockout mice, and has provided models for human genetic disease and the role of homeotic genes in development (1).The mechanism of HR has long been an elusive, yet fascinating, problem. Studies in prokaryotes and lower eukaryotes have provided much insight into the nature of this process, the recombination intermediates, the genes, and the proteins involved (2-4). The basic steps of HR are listed below together with the proteins required in Escherichia coli: (i) initiation of HR by a DNA double-strand break and͞or single-strand DNA formation by the RecBCD complex; (ii) exchange of DNA strands, including homology recognition and strand displacement, done by RecA-like proteins; (iii) heteroduplex extension, with branch or bubble migration, performed by RuvA plus RuvB or RecG to yield a recombination intermediate, a four-way DNA junction named the Holliday junction; and (iv) resolution of this heteroduplex Holliday junction by the endonuclease RuvC. Steps iii and iv have only very recently been elucidated: in E. coli the RuvA and RuvB proteins (encoded on one operon) have been shown to form a complex promoting ATP-dependent branch migration of Holliday junctions, a process of high importance for the formation of heteroduplex DNA. A second operon encodes RuvC and the orf-26 gene; RuvC is the endonuclease that binds specifi...

SummaryWe have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six-to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as ef®ciently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show signi®cant resistance to gammairradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.