Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMC s ) and͞or methyl methanesulfonate (MMS s ) and alterations in homologous recombination. Mutant xrs9 (Xray s , MMS s ) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xray s , MMC s ) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xray s , MMS s , MMC s ) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.Many similarities in DNA repair and recombination have been noted between plants, yeast, and higher eukaryotes. Meiotic recombination events in Arabidopsis thaliana (1) and maize (2) are unequally distributed along chromosomes, resulting in silent or hot spots of recombination, as in mammalian cells or yeast (3). In mammalian cells and yeast, high recombination frequency is confined to transcriptionally active regions, and this also has been concluded for maize (4). As in other organisms (5), the frequency of meiotic recombination in plants is influenced by environmental factors (6), sequence diversity in interspecific crosses (7), and the direction of crossing (8). In somatic cells, rates of homologous recombination can be stimulated by DNA-damaging treatments (9-11). This also has been observed in bacteria, yeast, and mammalian cells (12). However, a closer comparison reveals that plant cells are highly resistant to DNA-damaging treatments and are significantly more prone to the induction of increased recombination levels (9-11) than animal cells (13). This feature may be relevant for the late differentiation of germ cells in plants and the potential transmission of somatic recombination events to the germ-line (14, 15). Interestingly, genomic changes are influenced by environmental stresses (16,17), and specific induction of intrachromosomal homologous recombination by heat and salt stress has been documented (10, 11). Thus, regulation of genome stability during plant development is especially important. To help decipher enzymatic and regulatory aspects of DNA metabolism in plants, we isolated A. thaliana mutants hypersensitive to x-ray irradiation (xrs for x-ray sensitive), which also showed elevated sensitivity to DN...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 88%

Arabidopsis thaliana mutants altered in homologous recombination

Masson¹,

Paszkowski²

1997

“…Unfortunately, the number of events was so small that it is not possible to obtain good GT frequency estimates. Low frequencies of HR also have been reported when quantification was done for intrachromosomal recombination (ICR) in tobacco and Arabidopsis (14)(15)(16). Similarly, the rate of somatic crossover between homologous chromosomes was shown to be very low (17,18).…”

Section: ϫ5mentioning

confidence: 93%

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

Shalev

Sitrit

Avivi-Ragolski

et al. 1999

Self Cite

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...

“…In Table 1, only those kanamycin-resistant calli that proved to be the result of homologous recombination events are listed (see below). However, we also obtained calli (one for lines 1-7 and 1-12 each and nine for line [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] in which the kanamycin resistance was either due to illegitimate recombination events (molecular data not shown) or "escapes." Illegitimate recombination leading to kanamycin resistance may be explained by unexpected splicing of the intron sequence, whereas escapes may have arisen due to the relatively weak kanamycin selection, which had to be applied due to the relatively low level of resistance expressed by the artificial kanamycin construct (23).…”

Section: Methodsmentioning

confidence: 99%

“…However, several lines of indirect evidence indicate that transient DSBs enhance homologous recombination. Factors that introduce unspecific DSBs in DNA, such as x-rays (15,16) or methyl methanesulfonate (17), were shown to enhance intrachromosomal homologous recombination in plants. Excision of transposable elements was correlated with increased frequencies of recombination at the donor site between repeats flanking the element (18,19) or between repeats at ectopic sites (A.…”

mentioning

confidence: 99%

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Puchta

Dujon

Höhn

1996

361

283

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side ofthe DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation. (7), which has an 18-bp recognition site (8), was used to induce recombination reactions. In previous studies performed with I-Sce I, specific DSBs were induced in vivo in plasmid DNA transfected or injected into eukaryotic cells (9-12). Recently I-Sce I-mediated induction of genomic DSBs and their repair by homologous recombination were described for mouse cells (13,14). We demonstrated that expression of the I-Sce I gene led to specific DSBs in vivo in DNA molecules transfected into plant protoplasts (10). As a consequence extrachromosomal homologous recombination was induced. This reaction proceeds mainly via single-strand annealing and thus seems to be different from recombination reactions in the chromosome (as discussed in ref. 20). In the present communication we analyzed the mechanism by which DSBs at specific sites in the plant genome are repaired with homologous sequence information, and we tested whether induction of DSBs is correlated with an increase in recombination frequencies. MATERIALS AND METHODSCloning Procedures. Construction of the I-Sce I expression vector. The plasmid p35SISceI+ (10) containing an artificial I-Sce I open reading frame (ORF) under the control of the cauliflower 35S promoter and the cauliflower terminator was cut with BamHI and the ORF containing fragment was inserted in the unique BamHI site of the binary vector pCGN1589 (21) to result in the plasmid pCISceI.Construction of the target site. Into the EcoRV site of the plasmid pAF100 (22), the Xho I-Acc65I-cut, blunt-ended DNA fragment of the plasmid pAH5 (23) carrying the C-terminal half of an artificial, intron-containing kanamycin resistance gene was cloned....