JXB Advance Access originally published online on November 22, 2004
Journal of Experimental Botany 2005 56(409):1-14; doi:10.1093/jxb/eri025
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REVIEW ARTICLE |
The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution
Botany II, University of Karlsruhe, D-76128 Karlsruhe, Germany
* Fax: +49 721 6084874. E-mail: holger.puchta{at}bio.uka.de
Received 30 July 2004; Accepted 9 September 2004
| Abstract |
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The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A single-strand annealing mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative synthesis-dependent strand annealing mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.
Key words: Arabidopsis, deletion formation, gene conversion, homologous recombination, I-SceI, non-homologous end-joining, single-strand annealing, synthesis-dependent strand-annealing, tobacco
| Introduction |
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Double-strand breaks (DSBs) have to be eliminated before genomes can be replicated. Therefore, the repair of DSBs is critical for the survival of all organisms. Generally, DSBs can be repaired via two different pathways, either via homologous recombination (HR) or via non-homologous end-joining (NHEJ; also known as illegitimate recombination). Whereas in the former pathway the sequences are linked in regions that are identical to each other, in the latter the sequence information does not play a major role in the rejoining of the two double strands. An important question of recent research has been to find out under what conditions each pathway is used (for a general review see Paques and Haber, 1999
| Means to make ends |
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The prerequisite for the study of DSB repair is the production of DNA breaks in the plant nucleus at a given time point. In principle, DNA that has already been broken can be transferred into the nucleus or a break can be induced in vivo (e.g. by radiation, application of chemicals, during the replication of transposons, or by the use of restriction endonucleases). Plasmid molecules linearized by restriction enzymes in vitro (broken DNA) are used as a substrate for the transient transformation of plant cells. Transfer can be mediated by PEG transfection, electroporation or particle bombardment (for transfer techniques see Potrykus and Spangenberg, 1995
A classical way to induce DSBs is by the application of X-rays to the organism under study. Indeed irradiation has been used to induce mutations in plants for decades (e.g. in Arabidopsis: Redei and Li, 1969
; Shirley et al., 1992
). Using transgenic tobacco plants that carry intrachromosomal recombination substrates, it was demonstrated that the application of X-rays strongly enhances homologous recombination frequencies of the marker (Tovar and Lichtenstein, 1992
; Lebel et al., 1993
), a phenomenon that has been explained by the induction of DSBs into the marker DNA. However, one disadvantage of the use of X-rays is that, beside DNA breaks, other kinds of damage may be induced in the cell. In addition, although the number of breaks can be influenced by the application of different doses, it is not possible to influence where in the genome a break is induced. So in only some of the irradiated cells may a break occur at a specific locus. Thus, for a defined site the frequencies of recombination obtained by X-rays are many fold lower than with a site-specific break introduced uniquely at a precise location. For the induction of a site-specific DSB, transposable elements and rare cutting restriction endonucleases have been used in plants. DNA transposons are excised by the transposase out of the donor locus before integration somewhere else in the genome takes place. During this process the transient DSB is mainly repaired by NHEJ (Rinehart et al., 1997
). If homologous sequences are close to a transposon a strong increase in HR has been detected (Athma and Peterson, 1991
; Lowe et al., 1992
; Xiao and Peterson, 2000
). When a restriction site of a rare cutting nuclease like HO or I-SceI was included within the marker gene rather than a transposon, a similar enhancement of homologous recombination could be observed after expression of the enzyme, indicating that indeed the DSB is the inducing principal (Chiurazzi et al., 1996
; Siebert and Puchta, 2002
; Orel et al., 2003
). By regulation of expression of the respective transposase (in the case of a non-autonomous element), or endonuclease, the time point of break formation can be controlled. Expression can be achieved by transient transformation (Puchta, 1999a
) or by the control of a constitutive, organ-specific or inducible promoter after stable integration of the respective open reading frame into the plant genome (Schmidt-Puchta et al., 2004
).
| DSB repair by homologous recombination |
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The majority of studies on DSB repair in plants concentrate on homologous recombination. In part, this is because there is a general interest in HR for its application to genome manipulation (gene targeting), but also the consequence of DSBs having been demonstrated to be intermediates in meiotic recombination in yeast. In meiosis, DSBs are induced by the Spo11 protein, which is thought to originate from the subunit A of the archaebacterial topoisomerase VI. These breaks are repaired exclusively by HR using the sequence of the allelic chromosome as a matrix (for review see Keeney, 2001
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Ectopic recombination
In two independent studies, one based on DSB induction by the transposase of Ac, and the other by the endonuclease I-SceI, the question of how often an ectopic sequence can be used as the matrix for the repair of a DSB in plants was analysed (Shalev and Levy, 1997
This result was reminiscent of studies in which an I-SceI-induced genomic break was repaired by the use of homologous sequences on an incoming T-DNA from Agrobacterium tumefaciens (Puchta et al., 1996
). Integration of the T-DNA into the acceptor locus was mediated either by HR alone or by a combination of HR and NHEJ. As the same acceptor locus and the same donor sequences were used in both studies (T-DNA: Puchta et al., 1996
; ectopic DNA: Puchta, 1999b
), a comparison is possible and indicates that T-DNA is a better template for homologous DSB repair than an ectopic sequence. This is most probably due to the fact that the incoming T-DNA is more easily accessible for the copying processes than a chromosomal site. The original single-stranded T-DNA seems to be transformed to a double-stranded molecule in the nucleus (Tinland et al., 1994
; Tzfira et al., 2003
) and thus might be activated by the presence of factors involved in DNA repair or replication. Moreover, in contrast to a chromatin-packed ectopic locus, there might be fewer constraints of a steric nature for an interaction between the acceptor sequences and a free T-DNA.
The fact that a break can be repaired by a combination of HR and NHEJ clearly demonstrates that, in somatic cells, the classical double-strand break repair (DSBR) model of recombination (Szostak et al., 1983
) is not appropriate, as it predicts that both ends of the break would be repaired by HR in all cases. By contrast, other models such as the one-sided invasion (OSI) model (Belmaaza and Chartrand, 1994
) or the synthesis-dependent strand-annealing (SDSA) model of recombination (Formosa and Alberts, 1986
; Nassif et al., 1994
; Keeler and Gloor, 1997
) could explore the combination of HR and NHEJ on the repair of a DSB. However, whereas the OSI model could account for only the combination of HR and NHEJ, the SDSA model has the capacity to explain both kinds of events, repair by HR alone as well as by HR and NHEJ. To discriminate further between the models, experiments were performed with a T-DNA that carried homology to only one end of the break. The frequencies were compared with experiments in which a T-DNA was used harbouring homology to both ends of the break in the target locus (Fig. 2). According to the DSBR model the reaction should only proceed when homology to both ends of a break is present. The recombination frequencies obtained with the repair construct harbouring homology at both ends were only one-third higher than with the one-ended construct (Puchta, 1998
). Even this small difference could be explained by one-sided invasion of the unique second homologous end of the respective T-DNA. Hence, homology to only one end of the DSB is sufficient for an efficient homologous recombination reaction and DSB repair reactions in plant cells are initiated by one-sided initiation events. Using Ockham's razor (postulating the least number of entities required for the explanation of a phenomenon) the best suited model for repair of genomic DSB in somatic plant cells is the SDSA model (Fig. 3) as it is based on one-sided initiation and it is able to describe recombination events due to HR as well as due to a combination of HR and NHEJ events. The application of the SDSA model seems to be even broader (see below): during repair of breaks by NHEJ, in some cases sequences from elsewhere in the genome are copied into the break (Rubin and Levy, 1997
; Salomon and Puchta, 1998
; Gorbunova and Levy, 1999
). The respective junctions between broken DNA and inserted sequence are characterized by some base pairs (bp) of common homology, indicating a template switch, which is most easily explained by a SDSA mechanism (Fig. 4).
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Using the SDSA model as the central paradigm of somatic DSB repair, two other important predictions can be made: somatic DSB repair events between ectopic sequences are not accompanied by crossovers and, in gene targeting experiments, not only perfect integration events at the target locus by homologous recombination will be found. Indeed, since the early days of gene targeting in plants, recombinants were characterized in which only one end of the targeting vector integrated by homology (Risseeuw et al., 1995
The SDSA model describes double-strand break repair without formation of double Holliday junctions during the repair process. Consequently, this reaction will lead either to the non-homologous repair of one end of the break or in the case of perfect homologous recombination to the exclusive occurrence of gene conversion events not accompanied by crossovers (Fig. 3). Thus, due to their biological role, pathways of gene conversion at least partly differ in somatic and meiotic cells in higher eukaryotes. Whereas in meiosis double Holliday junctions occur (Schwacha and Kleckner, 1996
) and crossovers are a prerequisite for alterations in genetic coupling arrangements, in somatic cells the prevalence of gene conversion events avoids unpredictable deleterious genomic change by crossovers between ectopic sequences (Richardson et al., 1998
). This might be particularly important for preserving the stability of the plant genome, as many species contain ample amounts of repetitive DNA and somatic change can be transferred to the germ line (Walbot, 1996
).
Allelic recombination
During meiosis, DSBs are induced and repaired by the use of sequences from the homologue, resulting in either crossover or gene conversion. It was therefore important to test whether such a pathway is also operating in somatic cells. As gene targeting is not feasible for flowering plants it is, in contrast to mammalian (Moynahan and Jasin, 1997
) and yeast cells (Hiraoka et al., 2000
), not possible to manipulate directly allelic positions in plant genomes. To overcome this limitation a strategy was developed in which the transgene locus carrying the negative selectable marker cytosine deaminase (codA) with an I-SceI originally produced for the analysis of NHEJ (Salomon and Puchta, 1998
; see below) was rendered non-functional by the induction of a DSB repair reaction. Plants homozygous for the modified sequence were then crossed with plants homozygous for the original transgene. Thus, plant lines could be obtained with two different alleles at the transgene locus on homologous chromosomes. One allele contained an I-SceI site within the codA gene and the other either a deletion or an insertion within the region of the codA expression cassette that rendered both the I-SceI site and the codA gene non-functional. This approach was similar to the one of Dooner and Martinez-Ferrez (1997)
in which naturally occurring alleles of the bz gene of maize were combined. One of the alleles carried an active transposable element. However, the germinal excision of the element did not stimulate meiotic recombination, a fact that might be due to the programmed induction of DSBs at a specific time point during meiosis. After DSB induction by I-SceI expression selection was performed in heteroallelic tobacco plants for cells resistant to 5-fluorocytosine (5-FC) due to a non-functional codA gene. The occurrence of DSB-induced allelic recombination could be demonstrated (Gisler et al., 2002
). However, the vast majority of events resulted in a loss of function of the marker gene due to NHEJ. The frequency of allelic recombination was estimated to be about 104, similar to that of ectopic recombination obtained after DSB repair in tobacco by two different studies (see above; Shalev and Levy, 1997
; Puchta, 1999b
). Allelic recombination is induced by DSB at a specific site by at least three orders of magnitude. Thus, compared with meiosis, allelic HR is not a significant DSB repair pathway in somatic cells. As in these cells DSB repair occurs mainly by NHEJ, a process which might lead to major genomic alterations (see below), the inefficiency of allelic HR seems to be a prerequisite for the fixation of major changes in the genome. As somatic changes can be transferred to the germ line, this might be an important factor responsible for the enhanced plasticity postulated for plant genomes.
Intrachromosomal recombination
By contrast with the use of allelic and ectopic positions, sequences close to the break on the same chromosome or on the sister chromatid are more efficient matrixes for repair. Sister chromatids can be used as a template in the G2 stage of the cell cycle, and this pathway works efficiently in mammals (Johnson and Jasin, 2000
), however, due to experimental limitations it has not been possible to address this question in plant cells in connection with DSB induction until now (Molinier et al., 2004
). By contrast, a number of studies have been published on intrachromosomal DSB repair in plants (Athma and Peterson, 1991
; Chiruazzi et al., 1996
; Orel et al., 2003
; Siebert and Puchta, 2002
; Xiao and Peterson, 2000
; Xiao et al., 2000
) and a more detailed picture of the frequencies of repair as well as on the pathways has been determined. Depending on the structure of the respective chromosomal locus at least two different kinds of pathways may be used to repair the break. Whereas one pathway results in the formation of a deletion between direct repeats, the product of the other is a gene conversion event (Fishman-Lobell et al., 1992
). The deletion pathway is described by the single-strand annealing (SSA) model, whereas gene conversion can be explained by the synthesis-dependent strand-annealing (SDSA) model. The SSA model (Lin et al., 1984
, 1990
) describes a non-conservative reaction, resulting in a loss of information that is positioned between the annealing repeats (Fig. 3). SSA-like models have also been postulated to explain the loss of information during NHEJ (Nicolas et al., 1995
). As discussed previously the SDSA model is conservative, it describes transfer of information from a homologous donor sequence to the break site without loss of sequences at the donor locus (Fig. 3). The efficiency of the pathways can be addressed by using a different configuration of marker genes. According to the SDSA model the orientation of the donor sequence to the break has no influence on gene conversion and the break should lie within the homologous region. By contrast, constructs measuring the efficiency of the SSA mechanism should have a break site within two direct repeats. Using the respective markers in a recent study (Orel et al., 2003
) the frequencies of both pathways could be directly compared, indicating that the SSA pathway seems to be about five times more efficient than the gene conversion pathway.
However, of equal importance is to obtain an estimate of the number of breaks that are repaired in this situation by HR compared with NHEJ. In an attempt to determine this an assay was set up in tobacco based on interrupted overlapping halves of a ß-glucuronidase gene. Between the overlaps a negative selectable marker gene flanked by two I-SceI sites was inserted into the transgene (Fig. 5). As a consequence of I-SceI expression, after the excision of a negative selectable marker gene, a single genomic DSB had to be repaired. As the marker gene is lost, all resulting repair events (independent of their nature) could be isolated. It was demonstrated that the DSB was repaired in about one in three cases by HR. Thus, SSA is a highly efficient mechanism for DSB repair. SSA-like models can also be used to describe a class of NHEJ events (see below). Thus, SSA and SSA-like mechanisms might be the most prominent mode for the rejoining of broken DNA molecules in higher eukaryotes. If there are homologous sequences available in close proximity to the break they are regularly used for the annealing reaction, if they are not available, short patches of homology, which are present abundantly, might be used.
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The demonstration that, in plants, DSBs can be repaired efficiently by the use of nearby homologies seems also to be relevant for plant genome evolution: In the retroelement BARE-1 only a 742-fold excess of single long terminal repeats (LTRs) over its own internal regions has been found (Vicient et al., 1999
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If all experimental data are taken into account, a detailed picture can be obtained of how DSBs are repaired in somatic plant cells. The most efficient way to repair a break is by NHEJ (see below). If homologous sequences are available close to the break, the repair can take place in up to one-third of the cases by a single-strand annealing pathway. In about one out of 15 breaks, repair could also proceed via the SDSA pathway. This is two to three orders of magnitude more efficient than a gene conversion event that uses homology from an allelic or an ectopic site. DSB-induced gene conversion between members of tandemly arranged gene families might well play an important role in plant genome evolution. Various resistance genes are organized in tandem arrays in plant genomes and gene conversion events between the various members have been demonstrated by sequence analysis (Parniske et al., 1997
| DSB repair by non-homologous end joining |
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In higher eukaryotes, NHEJ seems to be the main mode of DSB repair (Sargent et al., 1997
But what are the molecular causes for the differences in DSB repair between Arabidopsis and tobacco? During DSB repair the size of a deletion depends on the processing of DNA ends. If broken ends are not religated directly the processing of such ends might result in the loss of DNA at the break site. Depending on the efficiency of DNA degradation more or less information will be lost. To address this question, particle bombardment was used to test the stability of linearized plasmid DNA in tobacco and Arabidopsis cells (Orel and Puchta, 2003
). Indeed indications were found that plasmid DNA is degraded to a lesser extent in tobacco than Arabidopsis. If a break was induced between promoter and open reading frame of a marker gene the expression level has reduced by a factor of two in Arabidopsis compared with tobacco. This observation was in contrast to the similar expression levels obtained for both species when the break was introduced a long distance from the marker gene. Whereas in the first case exonucleolytic digestion of few nucleotides would result in the destruction of the expression cassette, in the latter case the removal of 2 kb would be a prerequisite for the destruction of the ORF. Because endonucleolytic degradation would destroy all the information on a plasmid at the same rate, independent of a pre-existing break site, no differences should be found in the presence of such a nuclease activity. However, as the restoration of the marker ORF required a ligation step, the data of the expression assay alone do not suffice to discriminate between a more efficient exonucleolytic destruction of the marker gene or a less efficient ligation reaction in Arabidopsis cells. As a Southern blot analysis revealed differences in the degradation of linearized plasmid DNA between Arabidopsis and tobacco, it seems that most of the effect is due to a more efficient exonucleolytic degradation of DNA in Arabidopsis. However one question remains: What is the reason for the enhanced DNA degradation in Arabidopsis? This could be either due to less active DNA exonucleases or due to the better protection of DNA ends from degradation, for example, by DNA end binding factors (Liang et al., 1996
).
| Biotechnological applications: marker gene excision and site-specific integration |
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After the elucidation of the main mechanisms of DSB repair in plants it is of course an obvious step to use the induction of breaks at specific sites for the controlled manipulation of plant genomes. Two major applications have been developed: The excision of sequences from the genome and the integration of transgenes into specific sites of the genome.
Marker gene excision
Site-specific recombinases have been used efficiently for the excision of selectable marker genes (for a review see Hohn et al., 2001
; Hare and Chua, 2002
; Puchta 2003a
, b
). However, using this approach, recognition sites of the recombinases are left behind in the genome. Thus, for the performance of multiple genomic changes, a combination of different site-specific recombination systems has to be applied. The development of alternative approaches for site-specific alterations of genomes is of great interest in biotechnology. A very promising approach would be the combined elimination of the transgene sequences and the respective recognition sequence. Therefore, the use of highly specific restriction endonucleases to remove transgenic sequences from the plant genome is a useful alternative to the established site-specific techniques. The removal of the recognition site of the enzyme makes the process irreversible. It has been demonstrated that a marker gene can be efficiently eliminated from the plant genome either via HR or via NHEJ (including religation) by inducing DSBs by a rare cutting restriction enzyme (Fig. 5; Siebert and Puchta, 2002
). Thus, any sequence flanked by restriction sites could, in principle, be eliminated from the plant genome. In praxis, the open reading frame of the endonuclease, under an inducible promoter, could be included in the I-SceI cassette of the transgene to eliminate all superfluous sequence from the genome in one step (Puchta, 2003b
). The use of several other rare cutting endonucleases might also be useful for this purpose.
DSB-induced site-specific integration
Using co-transformation it could be demonstrated that different T-DNAs integrate into a single genomic locus (De Block and Debrouwer, 1991
; De Neve et al., 1997
). This indicates that a surplus of extrachromosomal DNA ends is recognized by the cell as breaks to be ligated, yielding a tandem arrangement of transgenes, which can then be linked to free genomic ends. It seems that the number of free genomic ends might be rate-limiting for the integration step of transgenes and indeed X-rays, which induce DSBs in the genome, can enhance transgene integration in plant cells (Köhler et al., 1989
). T-DNA integration into a DSB was demonstrated directly in a study on NHEJ using the codA gene harbouring an I-SceI site (see above; Salomon and Puchta, 1998
). In three out of four cases almost the entire sequence of the 2.4 kb long T-DNA was integrated. More detailed analysis on the frequencies of such events was performed recently using different site-specific recombinases. In approximately 1% of the transformants the T-DNA was found inserted into the break (Chilton and Que, 2003
; Tzfira et al., 2003
). Where I-SceI sites flanked the transformation marker on the T-DNA, integration of the DNA into the genomic I-SceI site by simple ligation could be demonstrated. This indicates that the T-DNA became double-stranded and was then cut, prior to integration, by the enzyme. These studies clearly demonstrate that DNA can integrate during DSB repair into the plant genome. Nevertheless, taking the frequencies, as well as the integration patterns, into account, it seems that DSB repair is not the only and not even the main mechanism of T-DNA integration. Various studies on T-DNA integration indicate that there are, in principle, two different classes of integration patterns (Fig. 7; Brunaud et al., 2002
). In most cases, the right border is conserved in the recombinants and contains no or very little microhomology to the target locus whereas longer homologies exist between the left border (combined with truncations) and the preinsertion site. In these events the two ends behave differently, and it has been postulated that integration occurs via single-strand annealing followed by the right border being linked to genomic sequences directly or indirectly by the VirD2 protein that is covalently bound to the 5' end of the T-strand (Tinland, 1996
). In a second class of events, not only the left but also the right border is partly truncated in the recombinants, and common nucleotides can be found in most cases at the junction site also between the right border and the preinsertion. This pattern of integration might be indicative for DSB repair. T-DNA molecules are transferred as single strands to the nucleus, where some of them become double-stranded before integration (Tinland et al., 1994
; Tzfira et al., 2003
). By interaction with break ends, a single- or double-stranded T-DNA could be copied into the genome via synthesis-dependent strand annealing. Alternatively, a double-stranded T-DNA could also be integrated at the break by two consecutive single-strand annealing reactions.
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Homologous integration into a genomic DSB was also achieved by the use of I-SceI. In up to 1% of the transformation events the target vector integrated into the homologous target locus (Puchta et al., 1996
Another gene targeting approach using DSB induction for the production of a recombination intermediate has been developed recently for Drosophila melanogaster by the group of Kent Golic (Rong and Golic, 2000
; Rong et al., 2002
). In this method the construct for targeting is integrated in the host genome flanked by two recognitions sites of a site-specific recombinase and includes a site for a rare cutting restriction endonuclease. By induced expression of the site-specific recombinase a DNA circle is excised from the genome. This circle is then linearized after the restriction enzyme (in this case I-SceI) has been expressed resulting in an activated DNA molecule with both ends homologous to the target sequence (for a simplified scheme see Fig. 8). In the female germline of Drosophila, gene targeting occurred in about one out of 500 cases. As gene targeting is a technique urgently needed in plant biotechnology there is considerable interest in determining whether the strategy is also applicable to plants (for discussion see also Kumar and Fladung, 2001
). On the one hand the set-up of the system seems to be very complex, as besides construction of a donor sequence with sites for a recombinase and a restriction enzyme, expression cassettes of both enzymes have to be included into the transgene construct or supplied in trans. On the other hand, if the reaction occurs in an efficient way in planta, every single seedling should represent an excision event. Thus, by the use of respective marker genes, large numbers of plants can be produced and easily screened.
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One has to see if and when the two new approaches, both based on the induction of DSBs, will help to solve the long-lasting problem of gene targeting in plants.
| Outlook |
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Over the last ten years a substantial body of information has accumulated relating to DSB repair in plants. The characterization of different pathways involved in DSB repair and the estimation of the respective frequencies makes it now possible for us to draw a quite detailed picture of the changes that occur during DSB repair in the plant genome. As sequenced genomes of an increasing number of plant species become available and, as a consequence, the evolutionary changes can be analysed at the whole genome level, current knowledge of DSB repair pathways will help to explain why genomes evolved in size and organization, resulting in the complexity that is found today. However, although major insights have been made, this picture of DSB repair in plant cells is far from complete, and neither questions on DSB-induced sister chromatid exchange nor on DSB-induced translocation have been addressed until now. A major challenge will be to refine techniques of genome manipulation further and this holds true particularly for the set-up of a reliable targeting technique. The induction of meiotic recombination by site-specific DSBs may be an important step along this path over the coming years.
| Acknowledgements |
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I would like to thank the different members of my group, especially Siegfried Salomon, Angela Kirik, Ralph Siebert, Brigit Gisler, Nadja Orel, Waltraud Schmidt-Puchta, Petra Oswald, and Christa Fricke, who over the years have carried out the work on DSB repair in plants, and the Deutsche Forschungsgemeinschaft and the European Union for funding my research in this area.
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