Towards the ideal GMP: Homologous recombination and marker gene excision

A mayor aim of biotechnology is the establishment of techniques for the precise manipulation of plant genomes. Two major efforts have been undertaken over the last dozen years, one to set up techniques for site-specific alteration of the plant genome via homologous recombination («gene targeting») and the other for the removal of selectable marker genes from transgenic plants. Unfortunately, despite multiple promising approaches that will be shortly described in this review no feasible gene targeting technique has been developed till now for crop plants. In contrast, several alternative procedures have been established successfully to remove selectable markers from plant genomes. Intriguingly besides techniques relying on transposons and site-specific recombinases, recent results indicate that homologous recombination might be a valuable alternative for the excision of marker genes

Marker-free transgenic plants

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance. Due mainly to consumer concerns, a suite of strategies (site-specific recombination, homologous recombination, transposition and co-transformation) have been developed to eliminate the marker gene from the nuclear or chloroplast genome after selection. Current efforts concentrate on systems where marker genes are eliminated efficiently soon after transformation. Alternatively, transgenic plants are produced by the use of marker genes that do not rely on antibiotic or herbicide resistance but instead promote regeneration after transformation. Here, the merits and shortcomings of different approaches and possible directions for their future development are discussed

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

What comparative genomics tells us about the evolution of the eukaryotic recombination machinery

The growing number of completely deciphered genomic sequences provides an enormous reservoir of data, which can be used for addressing questions related to functional and evolutionary biology. The wealth of this approach is documented by the fast growing numbers of recent publications in the field of evolutionary biology based on comparative genomics. Many proteins of the recombination machinery are conserved between plants, fungi and animals but some of them also show remarkable differences regarding their presence, copy number or molecular structure. For example, the protein responsible for double strand break (DSB) induction during meiosis, SPO11, which is related to the subunit A of the archaebacterial topoisomerase VI, is coded by a single gene in animals and fungi. In contrast, plants harbour three distantly related homologues, which seem to have non-redundant functions either in meiosis or in somatic cells and are indispensable for viability. Moreover, plants possess a homologue of the subunit B of the archaebacterial topoisomerase VI, not present in other eukaryotes. We also summarise the recent progress in the usage of genomic data to analyse the evolution of other DNA recombination factors. Finally, several recent studies report on a strong conservation of a reasonable number of intron positions between plants, animals and fungi. This kind of study provides a basis for comparative genomic analyses across kingdoms and demonstrates the existence of ancient introns, a topic of intensive debate

Differences in the processing of DNA ends in Arabidopsis and tobacco and its implication for genome evolution

Surprising species-specific differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks (DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum. In Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of the nature of the ends (blunt, 5\u27 or 3\u27 overhangs), linearized full-length DNA molecules were, on average, more stable in tobacco than in Arabidopsis. The relative expression of a β-glucuronidase gene encoded by the plasmid was similar in both plant species when the break was distant from the marker gene. However, if a DSB was introduced between the promoter and the open reading frame of the marker, transient expression was halved in Arabidopsis as compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the Arabidopsis genome is more than twenty times smaller than the tobacco genome

Biochemical Characterization of an Exonuclease from Arabidopsis thaliana Reveals Similarities to the DNA Exonuclease of the Human Werner Syndrome Protein

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRNexo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3\u27 -> 5\u27 direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3\u27-protruding strands. DNA with recessed 3\u27-PO4 and 3\u27-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3\u27-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg$^{2+}$ for activity, which can be replaced by Mn$^{2+}$. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants

The RTR complex as caretaker of genome stability and its unique meiotic function in plants

The RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RMI1 is involved in the processing of DNA recombination intermediates in all eukaryotes. In Arabidopsis thaliana the complex partners RECQ4A, topoisomerase 3α and RMI1 have been shown to be involved in DNA repair and in the suppression of homologous recombination in somatic cells. Interestingly, mutants of AtTOP3A and AtRMI1 are also sterile due to extensive chromosome breakage in meiosis I, a phenotype that seems to be specific for plants. Although both proteins are essential for meiotic recombination it is still elusive on what kind of intermediates they are acting on. Recent data indicate that the pattern of non-crossover (NCO)-associated meiotic gene conversion (GC) differs between plants and other eukaryotes, as less NCOs in comparison to crossovers (CO) could be detected in Arabidopsis. This indicates that NCOs happen either more rarely in plants or that the conversion tract length is significantly shorter than in other organisms. As the TOP3α/RMI1-mediated dissolution of recombination intermediates results exclusively in NCOs, we suggest that the peculiar GC pattern found in plants is connected to the unique role, members of the RTR complex play in plant meiosis