Stimulation of homologous recombination in plants by expression of the bacterial resolvase RuvC

Abstract

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...

High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene

“…The observed increase in gene-targeting frequency from 10 Ϫ4 to 10 Ϫ3 to between 10 Ϫ2 and 10 Ϫ1 makes it feasible to screen individual transformants for homologous insertion by using PCR. It might also be possible to further enhance gene-targeting frequency by stacking RAD54 with additional genes and mutations that enhance homologous recombination (30)(31)(32)(33). Gene targeting may be further enhanced by combining Rad54 expression with the induction of doublestrand breaks in DNA with zinc finger nucleases (34)(35)(36).…”

Re-engineering plant gene targeting

“…Both efforts have resulted in an increase in spontaneous homologous recombination, which might reflect either an increase in the efficiency of HR or an increase in the rate of induction of recombinogenic DNA damage. The fidelity of repair of a targeted (I-SceI-induced) break in tobacco lines overexpressing a nuclear-targeted RecA was characterized [51], and the frequency at which breaks were repaired entirely through HR was found to have increased. Interestingly, however, the frequency of gene replacement events did not increase.…”

“…These include the overexpression of E. coli RecA [24,50] and the expression of RuvC Holliday junction resolvase proteins [51]. Both efforts have resulted in an increase in spontaneous homologous recombination, which might reflect either an increase in the efficiency of HR or an increase in the rate of induction of recombinogenic DNA damage.…”

“…Double-strand break repair via homologous recombination. Arabidopsis homologs thought to be involved, based on mammalian models [51] are written in red. Proteins written in black are required for homologous recombination (HR) in some other organisms but obvious homologs do not exist in plants.…”

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

“…A key player in the E. coli recombination pathway is also the Holliday junction resolvase RuvC. Overexpression of this protein also enhances homologous recombination in plants (8). These data suggest that ectopic expression of several components of the homologous recombination pathway can stimulate the homologous recombination reaction.…”