Homologous Recombination and Gene Targeting in Plant Cells

Homologous Recombination and Gene Targeting in Plant Cells Bernd Reiss Max-Planck-Institut fu¨r Zuechtungsforschung, Carl-von-Linne-Weg 10, D-50829 Ko¨ln, Germany

Gene targeting has become an indispensable tool for functional genomics in yeast and mouse; however, this tool is still missing in plants. This review discusses the gene targeting problem in plants in the context of general knowledge on recombination and gene targeting. An overview on the history of gene targeting is followed by a general introduction to genetic recombination of bacteria, yeast, and vertebrates. This abridged discussion serves as a guide to the following sections, which cover plant-specific aspects of recombination assay systems, the mechanism of recombination, plant recombination genes, the relationship of recombination to the environment, approaches to stimulate homologous recombination and gene targeting, and a description of two plant systems, the moss Physcomitrella patens and the chloroplast, that naturally have high efficiencies of gene targeting. The review concludes with a discussion of alternatives to gene targeting. KEY WORDS: Nonhomologous recombination, Homologous recombination, Double-strand break repair, Recombination assay systems, Mechanisms of recombination, Recombination genes, Recombination mutants, Physcomitrella patens, Gene targeting in chloroplasts. ß 2003 Elsevier Inc.

I. Introduction Entire genomes are sequenced with amazing speed today and traditional methods are being increasingly replaced by DNA sequence-based approaches. As a consequence, the demand for reverse genetics tools is constantly increasing. Gene targeting is such a tool. Gene targeting, also referred to as targeted gene replacement, gene replacement, targeted homologous recombination, or International Review of Cytology, Vol. 228 0074-7696/03 $35.00

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Copyright 2003, Elsevier Inc. All rights reserved.

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‘‘the production of gene knockouts,’’ allows the precise modification of any gene in the genome. In this process, an in vitro modified copy is introduced into the genome by transformation to replace the endogenous gene by homologous recombination. The value of gene targeting lies in the precision of the process and the variety of its applications. Both make it an indispensable tool in many biological systems. One such application is the precise integration of transgenes at predetermined genomic positions. This technology gains increasing importance for the production of transgenic organisms and for human gene therapy. The main application, however, is analysis of gene function. Because gene targeting allows the production of specific and predictable changes in any chosen gene, it allows the systematic introduction of mutations and opens the door for novel approaches to analyze sequenced genomes. The power of this application is demonstrated best in budding yeast (Saccharomyces cerevisiae). In the absence of a replication origin, DNA with homology to the yeast genome almost exclusively integrates by homologous recombination. Moreover, eYcient gene targeting requires only minimal homology. Both together allowed the development of simple and highly eYcient methods for gene deletion. In one of them, gene replacement is achieved with nearly 100% eYciency using a crude polymerase chain reaction (PCR) for transformation of yeast cells (Baudin et al., 1993) after a short piece of DNA (30–50 nucleotides) homologous to the target gene was added by PCR to both ends of a selectable marker gene cassette (Wach et al., 1998). This technology allowed the functional characterization of nearly all of the about 6200 yeast open reading frames by systematic gene deletion and expression analysis (Winzeler et al., 1999). Such approaches would also be highly desirable for gene function analysis in plants in the future. Another example illustrating the value of gene targeting is modern mouse genetics. The complexity of the mouse has mostly prevented access to this organism by traditional genetics and the success of this model organism is largely based on gene targeting. The frequencies of gene targeting in mammalian cells were initially very low, but the systems were improved over the years and now allow the modification of nonselectable genes with high eYciencies (Muller, 1999; Sedivy and Dutriaux, 1999). Gene targeting in the mouse has nearly become a routine tool and the number of published mouse mutants has increased steadily resulting in more than 800 published knockout strains that had been generated by gene targeting in mouse embryonic stem (ES) cells. In contrast to the success in mouse ES cells and a few other systems (chicken DT40 cells, Trypanosoma brucei, Leishmania major, Drosophila melanogaster, and Dictyostelium discoideum), gene targeting remained problematic in other organisms. Prominent examples are somatic mammalian

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cells and the plant kingdom. Although gene targeting is possible in plants, an improvement of the eYciencies remained an unsolved problem.

II. Background A. History of Gene Targeting in Plants Paszkowski et al. demonstrated in 1988 that gene targeting is feasible in plants, shortly after the first knockouts in animal cells were published. These authors used polyethylene glycol (PEG)-mediated transformation of tobacco protoplasts and targeted an artificial model target locus that allowed direct scoring of gene targeting frequencies. However, the eYciencies were rather low (5
105 to 4
104). PEG-mediated transformation of protoplasts is relatively ineYcient and labor-intensive and therefore the possibility of using Agrobacterium-mediated transformation for gene targeting (Lee et al., 1990; OVringa et al., 1990) was perceived as a major breakthrough. The increase in transformation eYciency and the reduction in labor were expected to overcome the problem with the low gene targeting eYciencies. OVringa et al. (1990) also used an artificial target locus, but the repair construct was introduced by cocultivation of tobacco protoplasts. However, although larger numbers of transformants were generated and the gene targeting frequencies were comparable to those with PEG-mediated transformation, the problem was not solved. At that time, the use of artificial target loci was suspected of contributing to the low gene targeting frequencies. Lee et al. (1990) were the first to address this question using targeted modification of a natural, endogenous plant gene, the acetolactate synthase gene of tobacco. Targeted modifications of this gene confer resistance to the herbicide chlorsulfuron and therefore gene targeting events could be directly selected for after transformation of tobacco protoplasts by cocultivation with Agrobacterium. However, herbicide-resistant transformants were obtained with the same low frequency as with artificial loci before. Moreover, although the gene modifications were caused by homologous interactions with the target locus, the endogenous target gene remained unaltered and therefore no gene targeting was obtained. The low gene targeting frequencies are not unique to tobacco. Although the Arabidopsis thaliana genome (Wambutt et al., 2000) is relatively small and contains by far less repetitive DNA than tobacco, the gene targeting frequencies obtained in Arabidopsis were very similar (Halfter et al., 1992). Therefore, it appears unlikely that plants with complex genomes pose a special problem to gene targeting. Experiments that followed confirmed the low frequencies of gene targeting in plants, but also uncovered problems in its analysis. OVringa et al. (1993)

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observed products that looked like gene targeting at first glance, but turned out to result from random integration. In these events, homologous interactions between repair construct and target locus restored the repair construct, which then integrated at random into the genome. Such events were indistinguishable from real gene targeting with the commonly used analyses and therefore these results initiated more sophisticated and stringent strategies. These became standard in the future. The notoriously low frequency of gene targeting was essentially confirmed with a large number (more than 2 million) of transformants (Risseeuw et al., 1995). These studies also discovered that this process is often associated with additional rearrangements at the target locus. The low frequencies were not restricted to Agrobacteriummediated transformation since PEG-mediated transformation with an improved vector system also confirmed the low frequencies obtained earlier (Hrouda and Paszkowski, 1994). The same phenomena are also observed in lower plants. Gene targeting in the green alga Chlamydomonas is not more eYcient than in higher plants and it is also accompanied by additional rearrangements at the target locus (Smart and Selman, 1991; Sodeinde and Kindle, 1993; Gumpel et al., 1994). The higher plant targeting experiments described above were in tobacco or Arabidopsis leaf mesophyll protoplasts. Miao and Lam (1995) used another tissue, Arabidopsis root explants, and reported two targeting events in 2580 transformants. In addition, the use of a nonselectable endogenous gene and positive–negative selection was thought to have contributed to the eYciency. However, the material was never tested rigorously. Another endogenous gene was targeted with a similar eYciency (one gene targeting event in 750 transformants) shortly later (Kempin et al., 1997) using vacuum infiltration of Arabidopsis inflorescences (Bechtold et al., 1993). The gene targeting event was confirmed in this case. However, both publications (Miao and Lam, 1995; Kempin et al., 1997) reported single events. Therefore it is doubtful that their strategy consistently results in a high eYciency of gene targeting, especially since other natural loci could not be targeted with this eYciency (B. Reiss, unpublished observations). However, high (0.7 in 1000 transformants) gene targeting frequencies were recently obtained in Arabidopsis using single point mutations to convert an endogenous gene to a herbicide resistance gene and a significant number of events were tested rigorously (Hanin et al., 2001). In addition, high eYciency gene targeting was reported from rice using a new, highly stringent negative selection system (Terada et al., 2002). However, for both cases, future experiments have to demonstrate that the gene targeting strategies consistently work and can be applied to other loci. Various strategies to improve the eYciency of gene targeting were analyzed in other experiments. DNA damage caused by physical and chemical means was shown to stimulate homologous recombination, but this approach was never tested for gene targeting. In contrast, induction of double-strand

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breaks stimulates gene targeting 10- to 100-fold (Puchta et al., 1996). Unfortunately, however, the need for modification of the target locus prior to gene targeting largely prevented the application of this technology. Also positive–negative selection, allowing selection against random integration, was intensively analyzed in plants (Risseeuw et al., 1997; Thykjaer et al., 1997; Gallego et al., 1999; Wang et al., 2001), but, possibly with the exception of rice, did not solve the problem. The number of gene targeting events in these plants may generally have been too low to apply this strategy. Finally, another approach, overexpression of recombination enzymes (Reiss et al., 2000) and chimeric oligonucleotides (Beetham et al., 1999; Hohn and Puchta, 1999; Zhu et al., 1999, 2000), did not improve our ability to introduce targeted modifications into higher plants. In contrast, the moss Physcomitrella patens (Kammerer and Cove, 1996; Schaefer and Zryd, 1997) naturally has an outstandingly high eYciency of gene targeting and is the only plant that allows routine use of this technology.

B. Biological Basis of Gene Targeting Genetic recombination is a basic biological process found in all organisms. Recombination is involved in the mechanisms maintaining and safeguarding the genetic material. However, recombination also creates genetic diversity and actually may have an essential role in DNA replication. Recombination was studied intensively in bacteria and yeast and the models describing the recombination mechanisms in these organisms serve as a general guideline through this review. This section presents an abridged and simplified summary; a complete description would be outside the frame of this review, especially as recombination in bacteria (Eggleston and Kowalczykowski, 1991; West, 1992; Dunderdale and West, 1994; Kowalczykowski et al., 1994; Kowalczykowski and Eggleston, 1994; Camerini-Otero and Hsieh, 1995; Eggleston and West, 1996; Kowalczykowski, 1991, 2000) and yeast or vertebrates (Dunderdale and West, 1994; Camerini-Otero and Hsieh, 1995; Kleckner, 1996; Smith and Nicolas, 1998; Paques and Haber, 1999; Zickler and Kleckner, 1999; Flores-Rozas and Kolodner, 2000; Haber, 1999, 2000a–c; Sung et al., 2000; Gasior et al., 2001; Masson and West, 2001) is covered by other, excellent reviews. In addition, the literature used in this section can be found in these reviews. Generally, genetic recombination is divided into homologous and nonhomologous recombination. The hallmark of homologous recombination is the sequence-conservative exchange of information. Homologous recombination does not result in any loss or addition of sequence information. In addition, it requires extensive sequence homology between recombining partners. By contrast, nonhomologous recombination, often also referred to as

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illegitimate recombination, or nonhomologous end joining (NHEJ), is often accompanied by the loss or the addition of sequences and requires little or no homology between recombining partners. 1. Recombination in E. coli The basic concepts and the biochemistry of recombination were largely developed in Escherichia coli. At least 25 activities participate in genetic recombination in E. coli. These include strand-exchange proteins, resolvases, single-strand DNA-binding proteins, DNA polymerases, DNA topoisomerases, and DNA ligases. Homologous recombination in E. coli can been broken down into four steps: initiation, search for homology and strand exchange, branch migration, and resolution (see Fig. 1). The strand-exchange protein RecA has a central role in this process. After RecA has bound to single-stranded DNA, it promotes homology search, alignment of two molecules, and finally strand exchange. The substrate for RecA, single-stranded DNA, is provided mainly by the RecBCD helicase/nuclease and alternatively by the RecE and RecF pathway. The result of strand exchange by RecA is an intermediate in which the two double-stranded DNA molecules are connected to each other, a structure known as Holliday junction. RuvAB complex or RecG-mediated branch migration leads to an extension of the heteroduplex and the Holiday junction is finally resolved by the RuvC and RuvAB activities. Although RecBCD, RecE, and RecF might define discrete pathways, it now seems more likely that the use of these diVerent activities depends rather on the DNA substrate or other specific requirements of the recombination reaction. 2. Eukaryotic Recombination Genetic recombination in eukaryotes is far more complex than in bacteria and a distinction in meiotic and mitotic recombination further complicates matters. Recombination in yeast is by far best understood and most of the knowledge in eukaryotes is from this organism. Therefore the following discussion deals with the mechanisms and recombination models in yeast, unless otherwise mentioned. As for bacterial recombination, the entire process can be broken down into diVerent steps: initiation of recombination leading to a double-strand break, processing of the double-strand break, and, depending on the pathway, strand exchange or end joining (see Fig. 2). a. Initiation of Recombination Recombination in yeast is initiated by a double-strand break. These breaks may be induced by chemicals or radiation, or the induction is part of a developmental program, as in mating-type switching or meiosis. The induction of double-strand breaks in meiosis is a

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Initiation: RecBCD RecQ, RecJ, RecE

Strand exchange: RecA RecF, RecO, RecR, SSB

Branch migration: RuvAB RecG

Resolution: RuvC

FIG. 1 Simplified model for homologous recombination in E. coli. The entire process is broken down into the steps indicated. The first line below each step shows the E. coli proteins mainly involved in this reaction. The second line shows a selection of main accessory proteins or proteins involved in alternative pathways.

highly regulated process and requires a series of genes, most of which are meiosis specific. A key player is the SPO11 gene encoding a meiosis-specific endonuclease that contains several motifs of a novel family of type II topoisomerases. The activity of the SPO11 protein depends on several other genes; however, the role of these genes is not well defined yet (Smith and Nicolas, 1998; Paques and Haber, 1999). Homologues of SPO11 have also been found in several other organisms. The induction of double-strand breaks by HO endonuclease in mitotic recombination is yeast specific and will not be discussed here.

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Initiation SPO11

Processing XRS2 MRE11 RAD50

Diverging

KU70 DNA-PKcs

KU70

pathways

RAD52

RAD51

RPA

RAD55

RAD57 RAD54

non-homologous end joining

homologous recombination

FIG. 2 Simplified model for initiation of meiotic recombination in yeast. The main steps are described in the text. Proteins mediating interactions between steps are shown by shaded circles while other proteins are depicted as a shaded ellipse. The proteins involved in this process exceed by far the ones shown. The schema is reduced to essential and well-defined functions.

b. Processing of the Double-Strand Break Double-strand breaks are processed by a protein complex consisting of MRE11, RAD50, and XRS2. This complex converts the blunt ends left by SPO11 to 30 protruding singlestranded tails. The proteins in this complex have a dual role. They serve as a structural component and are involved, directly or indirectly, in the processing of the DNA ends. The MRE11 and RAD50 proteins show homology to the E. coli SbcD and SbcC proteins, respectively. Because SbcD has double-strand and single-strand nuclease activities, RAD50 might have a similar activity. The precise function of the complex is still unknown, however, because the MRE11, RAD50, and XRS2 genes are involved in many processes (DNA repair, homologous recombination, nonhomologous end joining, telomere maintenance, and double-strand break induction in meiosis), the proteins act in concert, and their biochemical functions are still

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not fully understood. However, the MRE11/RAD50 complex binds to and connects DNA ends and MRE11 stimulates nonhomologous end joining, suggesting that these proteins are more important for nonhomologous end joining than for homologous recombination (Paull, 2001). c. Diverging Pathways, Homologous and Nonhomologous Recombination Two main recombination pathways exist in eukaryotes, the homologous and the nonhomologous pathway. Currently it is thought that the interaction of the MRE11/RAD50/XRS2 complex with components of the homologous or nonhomologous end-joining pathways determines which pathway is used. The interaction with RAD52 will lead into the homologous pathway while an interaction with KU70/KU80 ends in nonhomologous end joining. Therefore, the fate of the processed intermediate will largely depend on the presence or absence of the activities participating in these pathways. The expression of genes involved in the homologous recombination or nonhomologous end-joining pathways is tightly regulated. In the mouse, KU70, a component of the nonhomologous pathway, is down-regulated in cells undergoing meiosis, presumably to avoid nonhomologous end joining and the resulting imprecise repair of double-strand breaks that are induced to initiate homologous recombination in meiosis (Goedecke et al., 1999). In addition, the homologous and nonhomologous pathways are tightly controlled in yeast (Haber, 2000a). d. Homologous Recombination The homologous recombination process seems to use diVerent mechanisms and therefore diVerent recombination models exist (for a complete description see Paques and Haber, 1999). These models are not mutually exclusive and the diVerent mechanisms they describe may all be used, depending on demand or other requirements of the process. The classic model is the double-strand break repair model of Szostak et al. (1983). This model is based on a strand invasion and a strandexchange reaction similar to the one mediated by RecA. Another one is the synthesis-dependent strand annealing model. According to this model, strand exchange involves the synthesis of new DNA and the return of the newly synthesized strands to the broken molecule followed by strand annealing. In the break-induced replication model, DNA synthesis continues by bubble migration for long stretches or a new replication fork is set up. The singlestrand annealing model is diVerent from the others since it does not involve strand invasion. In this model, single strands are formed by nuclease action and the heteroduplex is formed by annealing of complementary single strands. Genetic and biochemical studies in yeast have identified a series of proteins that participate in the homologous pathway, RAD51, RAD52, RAD54, RAD55, RAD57, RDH54, and RPA. Most of the corresponding genes belong to the RAD52 epistasis group. Because rad52 mutants are as sensitive

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to ionizing radiation alone as in combination with any of the other rad mutations (Game, 2000), RAD52 seems to have a prominent role. RAD52 is a DNA-binding protein with a higher aYnity for single- than for doublestranded DNA and binds to DNA as a ring-shaped multimer. RAD52 specifically binds to DNA ends and can interact with RAD51. This suggests that RAD52 functions by loading RAD51 to the ends of the processed double-strand break and thus this protein may initiate the strand-exchange process in homologous recombination. In addition, RAD52 assists RAD51 in interacting with another single-strand DNA-binding protein, replication protein A (RPA), an interaction necessary for RAD51-mediated strand exchange. However, the exact role of RAD52 is unclear and it also participates in another process, single-strand annealing. The central position of RAD52 may be unique to yeast (Sonoda et al., 2001). In contrast to yeast, rad52 mutants in mice are not hypersensitive to ionizing radiation. However, both yeast and humans have similar proteins that form similar structures and have similar biochemical activities. Therefore it is unlikely that RAD52 is not involved in homologous recombination in vertebrates, but it could be that vertebrates and yeast diVer in the mechanism they use for homologous recombination. Alternatively, other proteins, like the homologues of RAD52 found in yeast, could substitute for the RAD52 function or it is functionally replaced by a complex similar to that formed by RAD55 and RAD57. It is interesting to note that RAD52 does not have an obvious homologue in bacteria. It also seems to be absent in plants as no RAD52 gene was found in the sequence of the Arabidopsis genome, although homologues of most yeast recombination genes are present (B. Reiss, unpublished observations; for an analysis of the Arabidopsis genome, see Bevan et al., 2001). RAD51, the best studied eukaryotic recombination protein, is a homologue of the E. coli RecA protein. The two proteins are remarkably conserved in their amino acid sequence and both proteins form virtually identical nucleoprotein filaments on single-stranded DNA in the presence of ATP. Both proteins have DNA pairing and strand-exchange activities in vitro, but RAD51 is a relatively weak strand-exchange protein and needs accessory factors, such as the single-stand DNA-binding protein RPA. The similarity of RAD51 and RecA and the occurrence of RecA-like proteins in all organisms (Eisen and Hanawalt, 1999) suggest that RecA-like strand-exchange reactions also have an important role in homologous recombination in eukaryotes. The yeast genome contains further proteins with homology to RecA or RAD51. The best known example is DMC1, a meiosis-specific recA-like gene. DMC1 has significant homology to RAD51 and the two proteins have some biochemical activities in common, but diverge in others. Other examples of yeast proteins with homology to RecA or RAD51 are RAD55

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and RAD57. These proteins show limited homology to RecA or RAD51. The proteins form a heterodimeric complex and interact with RAD51. In analogy to the RecO and RecR proteins of E. coli, this complex may help to load RAD51 onto single-stranded DNA under conditions in which RAD51 is in competition with RPA. Eukaryotic genomes contain even more genes with limited homology to RAD51. These are generally referred to as RAD51-like genes (Shinohara and Ogawa, 1999; Thacker, 1999) and currently include XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D. Some of these genes were renamed and published under diVerent names. The corresponding proteins, including RAD51, may form functional complexes. These complexes have essential functions in homologous recombination since the proteins interact with each other or RAD51 and RAD51-like genes are necessary for eYcient homologous recombination (Sonoda et al., 2001). In contrast to yeast, RAD51 gene function is essential in vertebrates (Thacker, 1999; Sonoda et al., 2001). In the mouse, homozygosity in an RAD51 defect results in embryonic lethality and also cultured cells are not viable. Similarly, chicken DT40 cells deficient in RAD51 are nonviable and the defect results in randomly distributed chromosomal breaks in conditionally deficient cells. The diVerence between yeast and vertebrate cells may lay in the diVerent sizes or complexities of the genomes, but it is more attractive to think that RAD51 genes have acquired an additional role in higher eukaryotes. DT40 cells depleted of RAD51 arrest in G2–M, which suggests that the defect is more in the regulation of the cell cycle than in recombination. Moreover, mammalian RAD51 interacts with the cell cycle or apoptosis-related proteins such as p53, BRCA1, and BRCA2. e. Nonhomologous End Joining Nonhomologous recombination is far less understood than homologous recombination. Nevertheless, nonhomologous end joining is an important pathway in vertebrate cells and yeast (Haber, 2000a,c; Sonoda et al., 2001). Mammalian and yeast cells are remarkably similar in their choice of pathways. Double-strand breaks induced by sitespecific endonucleases are repaired to 30–50% in mammalian cells and to 70% in yeast by homologous recombination. The remainder is repaired by nonhomologous recombination. In yeast, the dominant process is precise religation of the ends, but if religation is prevented, the ends are modified. These modifications consist of deletions of a few to a few thousand base pairs. The majority of the junctions show homologies of 1–5 base pairs between the interacting partners, or consist of insertions of a few base pairs that originate from misalignments and filling in of the overhanging ends, very similar to the modifications observed in nonhomologous end joining in mammalian and plant cells. In yeast, direct back-ligation as well as ligation after end modification depend on the yeast homologues of KU70 and

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KU80. In addition, the RAD50, MRE11, XRS2, DNA ligase IV, and the yeast XRCC4 homologues are involved. However, additional pathways must exist in yeast and in vertebrate cells as nonhomologous end joining is not abolished by ku70 mutations in both organisms (Haber, 2000c; Vasquez et al., 2001). f. Links to Mismatch Repair DNA mismatch repair (Paques and Haber, 1999; Evans and Alani, 2000; Hsieh, 2001) plays an essential role in the maintenance of genetic information. The main function of mismatch repair is thought to be the correction of postreplication errors since the system mainly recognizes base exchanges and short DNA insertions or deletions. The E. coli mismatch repair system consists of three components, MutH, MutL, and MutS. The MutS protein binds to DNA mismatches as a homodimeric complex and recruits MutL. This activates the MutH endonuclease that cleaves the newly synthesized DNA. Mismatch repair in eukaryotes appears far more complex, but homologies to the E. coli system exist. Several diVerent MSH proteins, the homologues of MutS, are present in eukaryotes. The proteins form heterodimers that have specialized functions in mismatch recognition. The MSH2–MSH6 heterodimer is specialized for base–base mismatches while both MSH2–MSH6 and MSH2 and MSH3 participate in the repair of small insertions. In addition, homologues of the MutL protein (MLH) exist, but no MutH homologue has been identified so far. The mismatch repair system also plays a role in recombination. Annealing of the DNA strands leads to a heteroduplex. Homeologous sequences, i.e., DNA sequences containing a small number of mismatches, recombine less eYciently than homologous sequences. This diVerence is lost in mismatch repair mutants and therefore the mismatch repair system is involved in the control of which molecules recombine. In addition, the system can lead to the rejection of molecules with insuYcient homology. Mismatch repair plays a major role in gene transfer between closely related species, the establishment of genetic barriers, and might help to prevent recombination between repeats or members of gene families sharing sequence homology. The exact role of the mismatch repair system in recombination remains to be determined, but MSH2 plays a global role in the rejection and other genes exist that have more specialized functions (Evans and Alani, 2000). Some mismatch repair proteins play a direct role in recombination. In yeast, the MSH2–MSH3 complex cooperates with the nucleotide excision repair proteins RAD1 and RAD10 to remove unpaired single strands at the ends of the annealed region after single-strand annealing or strand invasion. In addition, MSH4 and MSH5, two MutS homologues with no apparent role in mismatch repair, and MLH1, which plays a role in mismatch repair, are involved in the modulation of crossover frequency.

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g. Connections to Gene Targeting In contrast to natural recombination processes, one of the recombination partners is artificial and introduced by transformation in gene targeting. The integration of transformed DNA is thought to follow preexisting recombination pathways. Therefore, the predominance of random integration observed in mammalian or plant cells is believed to indicate an active nonhomologous pathway in these cells. In contrast, the high eYciency of gene targeting in yeast is thought to reflect the dominance of the homologous pathway in this organism. However, as discussed previously, both pathways are used with comparable eYciencies in yeast and mammalian cells to repair a genomic double-strand break. This apparent contradiction may be related to diVerences in the use of recombination pathways (Sonoda et al., 2001). In yeast, defects in the homologous, but not in the nonhomologous, pathway cause sensitivity to ionizing radiation and haploid yeast prefers the homologous pathway except for G1 when no obvious homologous partner for recombination exists. Therefore, the nonhomologous end-joining pathway may serve as a backup system in yeast, in case the homologous pathway is not functional. In contrast, mouse ES cells deficient in either pathway are equally sensitive to radiation. However, adult mice defective in the nonhomologous end-joining, but not in the homologous pathway, are hypersensitive to radiation. Therefore proliferating mouse cells seem to use both pathways equally, but the nonhomologous route is preferred in nonproliferating cells. These observations correlate with the frequencies of gene targeting observed in these systems and suggest that the homologous pathway is less preferred in diVerentiated vertebrate cells. However, details of the recombination processes itself may also contribute to the diVerences between yeast and vertebrates. Although chromosomal homologues may be used in yeast, such events are rare in vertebrate cells and recombination seems to be restricted to the sister chromatid, even after induction of a double-strand break. This diVerence in interhomologue recombination might indicate that mitotic vertebrate cells, in contrast to yeast, have developed mechanisms to restrict recombination to a particular partner. Furthermore, homologous recombination in vertebrate cells seems to be suppressed in G0/1 since key homologous recombination genes are induced by DNA damage in this stage of the cell cycle in yeast, but not in vertebrate cells. These observations suggest that yeast and vertebrate cells diVer in important aspects of regulation of recombination and these might account for the diVerences observed between double-strand break repair and gene targeting eYciencies. The gene targeting process itself is poorly understood. The gene targeting problem is highlighted in a model experiment in yeast (Haber, 2000c) in which a fragment carrying a marker gene was released from the genome using a site-specific endonuclease. Although the double-strand break created by the endonuclease was repaired with high eYciency (90%), targeting of a

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mutated marker gene also present on the chromosome was below 0.1%. This low eYciency indicates a strong bias to correct the heteroduplex to the resident DNA. Mismatch repair is important for the rejection process as this bias depended on the presence of this system. The diVerence between naturally occurring recombination processes and gene targeting might be that transformed DNA does not resemble authentic recombination substrates. In addition, in gene targeting the DNA actually may not integrate into the genome by crossovers in the regions of homology. It also could be that the free ends of the integrating fragment set up replication forks after strand invasion and start to copy portions of the target locus (Haber, 2000a), a model that perfectly explains the ectopic events often observed in gene targeting experiments.

C. Recombination and Gene Targeting Assay Systems in Plants 1. Recombination Assay Systems Cellular survival to DNA damage was frequently used to analyze genetic recombination. Recombination participates in the repair of DNA damage that aVects both strands. Double-strand breaks induced by ionizing radiation such as X- and gamma-rays and the radio-mimicking agent methyl methanesulfonate (MMS) are the best known examples. However, mitomycin C also causes lesions that aVect both strands. Historically, the rad mutants in yeast were classified using sensitivity to ionizing radiation caused by X-rays (Game, 2000). Sensitivity to ionizing radiation (Davies et al., 1994; Jiang et al., 1997; Masson et al., 1997; Masson and Paszkowski, 1997), MMS (Mengiste et al., 1999), and mitomycin C (Reiss et al., 1996; Masson and Paszkowski, 1997; Mengiste et al., 1999) was also used in plants to isolate and classify recombination mutants and to analyze homologous recombination. Sister chromatid exchange and twin sector formation, both naturally occurring processes, were also used to analyze homologous recombination. Sister chromatid exchange (Reiss et al., 2000) directly scores the crossover events leading to the exchange of portions of sister chromatids using visual inspection after staining of chromosomes. The frequency of the counted chiasmata directly reflects the activity of recombination in this assay. Whereas sister chromatid exchange examines events between chromatids of the same allele, the twin sector formation assay at the sulfur gene locus (Shalev et al., 1999; Gorbunova et al., 2000) addresses interhomologue recombination. The sulfur gene controls chlorophyll pigmentation in tobacco and spontaneous recombination in a heterozygous background leads to the formation of easily detectable sectors in leaves. The heterozygous plants are pale green and viable. Recombination at this locus leads to the

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formation of homozygous, pale white or dark green cells. Thus somatic recombination events are visible as dark green and white sectors in the pale green background of heterozygous individuals. The more common assay systems use artificial recombination reporter genes. These consist of a reporter gene that was split in two, complementary, nonfunctional parts. The half genes have overlapping regions of identical sequence that allow the regeneration of a functional reporter by homologous recombination. The reporter genes initially used were antibiotic resistance genes such as the neomycin phosphotransferase II (nptII ) (Baur et al., 1990; OVringa et al., 1990; De Groot et al., 1992; Hrouda and Paszkowski, 1994) and the hygromycin phosphotransferase gene (Bilang et al., 1992). These were replaced by the visually detectable reporter gene b-glucuronidase (GUS) (Lyznik et al., 1991; Puchta and Hohn, 1991a,b; Shalev et al., 1999) in systems developed later. The advantage of these systems is that GUS expression can be detected using a highly sensitive enzymatic assay as well as by staining tissues in whole plants. The recombination assays using artificial recombination reporter genes are based on two diVerent principles. The two complementary half genes are localized on separate molecules in the extrachromosomal recombination systems and these are introduced together into the plant cell by transformation. The functional reporter genes are likely to be created in a recombination reaction that occurs prior to chromosomal integration of the interacting molecules. In the systems using antibiotic resistance genes, transformants are regenerated and the recombination frequencies calculated from the number of resistant transformants. This regeneration and selection step requires that the recombined sequences have been integrated into the genome, a process that usually occurs by random integration. Therefore these recombination assays are more indirect and any feature that influences DNA integration may distort calculated recombination frequencies. Recombination assays using intrachromosomal recombination are by far the most widespread systems. The split reporter gene in these systems is located on one single molecule and forms an artificial repeat. The half genes are placed next to each other in either direct or inverted orientation and are separated by a spacer region. The spacer usually contains an independent selectable marker gene that is used to select for the integrated construct in the transformation step. Homologous recombination between the half genes forms a functional reporter that can be selected for or visualized by staining. Depending on the relative orientation of the repeats, the internal spacer region is either deleted or inverted in this process. Systems using the kanamycin resistance gene (Peterhans et al., 1990; Assaad and Signer, 1992; Tovar and Lichtenstein, 1992) and the GUS gene as a visual marker are available (Swoboda et al., 1994). In addition, the cauliflower mosaic virus was used to monitor intrachromosomal recombination after

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engineering an artificial repeat derived from two distinguishable virus strains (Gal et al., 1991; Swoboda et al., 1993). Recombination assay systems using antibiotic resistance genes require the regeneration of single cells that have acquired the resistance. Regeneration of transformed tissue involves tissue culture, a procedure known to induce mutations. Moreover, tissue culture conditions may lead to alterations in the recombination processes and thus aVect recombination frequencies. Therefore, these systems are biased by a variety of parameters. In contrast, systems using the GUS recombination reporter gene rely exclusively on reporter gene expression and therefore are more direct. In addition, GUS allows whole plant recombination assays and the recombination frequencies can be directly obtained from the number of GUS-positive cells. Nevertheless, factors that influence reporter gene expression and the well-known technical problems in GUS staining might aVect the results as well. Although intrachromosomal recombination has mainly been used in plants as a tool to investigate recombination, this process may also be useful to remove selectable marker genes. A marker gene flanked by attP regions was deleted with amazingly high eYciency from a transgenic plant line by intrachromosomal recombination (Zubko et al., 2000). However, this extraordinary high frequency of intrachromosomal recombination remained unexplained and further work is needed to understand the underlying principle (Puchta, 2000; Hohn et al., 2001). 2. Gene Targeting Assay Systems The hallmark of a gene targeting assay system is a feature for easy detection and quantification of gene targeting events. Therefore, most systems used artificial model loci. The target loci were designed such that a gene targeting event converts a defective gene into an active marker, which confers resistance to antibiotics or herbicides. Because a dominant trait is generated, the gene targeting frequency can be directly calculated from the number of transformants. As with the other recombination assays described before, most systems used split antibiotic resistance genes with complementary and overlapping regions. One of the split genes is placed into the genome by PEG or Agrobacterium-mediated transformation and the other one is part of the repair construct used for the gene targeting experiment. In addition, in most cases the repair construct also carries an independent selectable marker gene that is used to determine transformation eYciencies. The recombination reporter genes mostly used for this purpose were the antibiotic resistance genes hygromycin phosphotransferase (Halfter et al., 1992) and nptII (Paszkowski et al., 1988; OVringa et al., 1990; Hrouda and Paszkowski, 1994). In a variant of this set-up, an nptII gene exclusively expressed in seeds was converted into a constitutive gene by targeting a CaMV 35S

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promoter to a seed-specific promoter (Reiss et al., 2000). Herbicide resistance genes, also used as recombination reporters for gene targeting, are special. The herbicide-sensitive variants of these genes are naturally occurring genes and therefore are already present in the genome. The clear advantage of systems based on these genes is that they use natural loci as targets. In addition, there is no need to generate a transgenic reporter gene line by transformation since the target genes are already present in the genome. The acetolactate synthase gene was the first herbicide resistance gene used for this purpose. A single amino acid exchange in the acetolactate synthase protein of tobacco confers resistance to the herbicide chlorsulforon. To analyze gene targeting in tobacco, a defective, chlorsulforon-resistant acetolactate synthase gene was used that creates a functional resistance gene by homologous recombination (Lee et al., 1990). The protoporphyrinogen oxidase gene is another herbicide resistance gene that was used to analyze gene targeting in Arabidopsis (Hanin et al., 2001). This gene has the advantage that more than one mutation is necessary to create a resistant variant, a feature that considerably reduces background by spontaneous mutations. In addition, a variety of endogenous, nonselectable genes were used in gene targeting experiments and pooling, and PCR-based strategies without (Kempin et al., 1997) or with negative selection (Miao and Lam, 1995; Thykjaer et al., 1997; Gallego et al., 1999; Terada et al., 2002) were used to identify the targeting event. The use of transgenic model target loci, as opposed to natural genes, for gene targeting assay systems is problematic. The transgenes integrate at random and therefore there is no control over the integration site in the genome. However, the chromosomal environment around the integration site can aVect recombination. The activity of recombination is not evenly distributed over the chromosome as suggested by the variability of gene targeting frequencies in mouse ES cells, ranging from 80% to less than 1 in 1000, the occurrence of hot and cold spots of recombination, and the limited colinearity of physical and genetic maps. Therefore, insertions in relatively inactive regions may cause low gene targeting frequencies at that locus. In addition, the activity of the transgene may be influenced by a variety of factors. Transgenes are often inserted in multiple copies and they are often rearranged further. Both can lead to gene silencing or render the transgenes nonfunctional. Because the activity of the recombination reporter gene can be verified only by gene targeting, a particular line carrying such a system remains problematic until gene targeting has been successfully demonstrated. Two types of vectors, generally referred to as targeting vector or repair construct, are used for gene targeting, replacement, and insertion type vectors (for a complete description see Morton and Hooykaas, 1995; Muller, 1999). Both contain a cloned copy of the chromosomal target gene that can be derived from either genomic DNA or cDNA. The replacement

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vector carries the selectable marker gene within this piece of DNA. This marker also serves to disrupt the target locus. The insert is released before transformation as a DNA fragment colinear with the target gene, except that it contains the selectable marker gene. Because the regions of homology face outward from the selectable marker gene toward the ends of the vector, vectors of this type are also called ends out vectors. Replacement vectors cause the replacement of target locus sequences by vector sequences in a double crossover-like process within the sequences flanking the selectable marker gene. Because the replacement takes place in a single step, these vectors are preferred for gene targeting. In contrast, in insertion-type vectors the entire vector is integrated into the genome by a single crossover. In this vector type, cloned target locus sequences, vector, and selectable marker gene are next to each other and the vectors are either used as circular molecules or are linearized within the region of homology. Because the entire piece of vector DNA integrates into the genome, the target gene is partially duplicated, a fact commonly regarded as a major drawback of this type of vector.

III. Mechanisms of Recombination and Gene Targeting in Plants A. Extrachromosomal Recombination Extrachromosomal recombination, sometimes also referred to as intermolecular homologous recombination, served as an important model to study the mechanisms of recombination at the beginning when other methods were not yet available. This system has been used to analyze a variety of basic parameters in tobacco or Nicotiana plumbaginifolia plants (Puchta and Meyer, 1994; Puchta et al., 1994; Puchta and Hohn, 1996). Extrachromosomal recombination is a naturally occurring process. Examples are recombination between viruses or the genetic exchange between T-DNAs, which can occur during Agrobacterium-mediated transformation (OVringa et al., 1990; Tinland et al., 1994). However, for recombination studies in plants, assay systems were developed that are based on artificial recombination substrates and these are introduced into protoplasts by DNA transformation. In general, extrachromosomal recombination is much more eYcient as gene targeting or intrachromosomal recombination, a feature that explains the popularity of these systems. The first application of extrachromosomal recombination was the analysis of how homology aVects the eYciency of homologous recombination. Using an assay system based on the antibiotic resistance gene nptII, Baur et al. (1990) found that a homology of as little as 53 bp was suYcient for recombination, but the eYciency increased with increasing homology. Using a diVerent assay

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system, GUS, however, a homology below 456 bp was found to be insuYcient while frequencies above this threshold were comparable to the previous data (Puchta and Hohn, 1991b). The eYciency of recombination is substantially influenced by the topology of the recombination substrates. Circular plasmids or a combination of circular and linear plasmids recombine much less eYciently than linearized plasmids or single-stranded DNA molecules with complementary sequence (Bilang et al., 1992). Supporting the importance of free DNA ends for the recombination process, double-stand breaks introduced in vivo into circular molecules enhanced extrachromosomal homologous recombination (Puchta et al., 1993). The relationship between extrachromosomal and chromosomal recombination is not clear. Extrachromosomal recombination is a precise (Hrouda and Paszkowski, 1994), fast (Puchta et al., 1992), and genuine recombination process that involves the exchange of flanking markers (Puchta and Hohn, 1991a). The mechanism appears to involve the generation of single-stranded DNA molecules and annealing of complementary strands ends and therefore a single-strand annealing model was suggested for extrachromosomal recombination in plants (Puchta and Hohn, 1991a; De Groot et al., 1992). Some of these features are also observed in chromosomal recombination and, as discussed in the next section, single-strand annealing also plays a role in chromosomal recombination. However, the mechanisms of chromosomal recombination are more diverse and extrachromosomal recombination is not likely to reflect all facets of this process. Moreover, the eYciencies of these two processes diVer dramatically, probably because the number and nature of the molecules involved are fundamentally diVerent. Most importantly, chromatin is unlikely to play a role in extrachromosomal recombination while it has a function in chromosomal recombination. As in other plants before, the eYciency of extrachromosomal recombination in maize depends on the length of homology and the topology of the recombination substrates (Lyznik et al., 1991). However, in contrast to animal cells, short, nonhomologous sequences at the 30 -end of the substrates inhibited recombination. In addition, some aspects of extrachromosomal recombination could be plant species specific. The mechanism of extrachromosomal recombination may be diVerent in Petunia since single-strand annealing is less important than other recombination mechanisms (Engels and Meyer, 1992). Gorbunova and Levy (1997) have used extrachromosomal recombination to study the mechanisms of nonhomologous end joining in tobacco cells. Analysis of the junctions between recombined plasmids showed that the ends were rarely conserved. Instead, most of them consisted of deletions at both ends and rejoining occurred at short repeats. In addition, a number of junctions contained insertions of foreign DNA, originating either from internal portions of the plasmids or from the tobacco genome. Therefore,

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intensive DNA degradation and synthesis are involved in the repair of a double-strand break by nonhomologous end joining in plants.

B. Chromosomal Recombination To study the repair of a double-strand break in the chromosome, Salomon and Puchta (1998) placed a recognition site for the rare cutting endonuclease I-SceI into a negative selection marker and inserted this transgene into the tobacco genome. Transient expression of I-SceI and negative selection yielded events that used repair of the double-strand break by nonhomologous end joining. The junctions of such events consisted either of deletions at the ends without further modifications or the deletions contained insertions. In both cases, microhomologies of a few nucleotides were involved in the ligation of the ends. The inserted sequences were usually short, but elements of the deleted transgenic locus, chromosomal tobacco DNA, and an insertion of almost the entire T-DNA used to express the endonuclease were also found. The nature of recombination products, especially the observation of microhomologies, suggests that chromosomal double-strand breaks, like the plasmid ends in extrachromosomal recombination, are repaired by single-strand annealing or synthesis-dependent strand annealing (Salomon and Puchta, 1998). Nonhomologous end joining may be promoted by palindromic and AþT-rich sequences, elements that preferentially create or stabilize single-stranded DNA, since such elements are overproportionally represented in the junctions (Muller et al., 1999). The mechanism of double-strand break repair may be important for genome evolution in plants. Insertion of sequences into double-strand breaks could be a new homology-independent mechanism to copy genomic DNA to new positions (Salomon and Puchta, 1998). Moreover, diVerences in the mechanism of double-strand break repair may account for the diVerences in genome sizes between plant species. The genome sizes of Arabidopsis and tobacco, two species with a 20-fold diVerence in genome size, were inversely correlated with the size of the deletions occurring in these species (Kirik et al., 2000). The deletions in Arabidopsis, the plant with the smaller genome, were significantly larger than in tobacco and insertions of filler sequences were entirely lacking. Therefore, this process tends to result in loss of DNA in plants with small genomes while the reverse happens in organisms with large genomes. Indeed, illegitimate recombination was shown to be the driving force for the reduction of the size of the Arabidopsis genome (Devos et al., 2002). Recombination hot spots are chromosomal regions with a high activity of recombination. The hot spots in yeast are precisely defined positions at which meiotic recombination is initiated by a double-strand break. Recombination hot spots are also observed in maize. However, meiotic recombination at

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such a hot spot in maize, the bronze gene, is induced randomly (Dooner and Martinez-Ferez, 1997b). In addition, the induction of double-strand breaks by excision of the transposable element Ac present in the bronze gene did not enhance the frequency of meiotic recombination at bronze, except when the gene was present as a tandem duplication (Dooner and Martinez-Ferez, 1997a). Intrachromosomal homologous recombination at an artificial repeat was stimulated by Ac/Ds more than 1000-fold in Arabidopsis (Xiao and Peterson, 2000) and significantly between the direct repeats flanking the p1 gene coding region in maize (Xiao et al., 2000). The frequency of recombination is not equally distributed across the genome. In Petunia, recombination in the central region of chromosome II is generally suppressed and certain regions of chromosome seem to be more open for the integration of DNA since the distribution of T-DNA insertions along the chromosome was not even (ten Hoopen et al., 1996). Recombination at ectopic positions, nonallelic sequences in the genome that share sequence homology, occurs with a frequency comparable to allelic recombination (Gisler et al., 2002). Both processes are rare, even after the induction of a double-strand break (Shalev and Levy, 1997; Puchta, 1999), and use comparable mechanisms for double-strand break repair (Puchta, 1999). Also unequal crossing-over in meiosis, a process similar to ectopic recombination, is rare in plants (Jelesko et al., 1999).

C. Targeted DNA Insertion Gene targeting experiments often yield unexpected products in addition to, or instead of, the expected modification of the target gene. Such a product, a modified repair construct, was obtained by OVringa et al. (1993) in a gene targeting experiment using Agrobacterium-mediated transformation. Gene replacement at the target locus and gene conversion at the repair construct using target locus sequences are the predicted, reciprocal outcomes of a homologous interaction between target locus and repair construct sequences. However, while in gene conversion target locus sequences are copied into the repair construct using homology at both sides of the molecule, the molecule described by OVringa et al. (1993) was generated by nonreciprocal homologous recombination. Moreover, a large portion of the genomic region flanking the target locus, extending far beyond the region of homology with the repair construct, was copied, this molecule was rearranged further, and then inserted at a random position. Therefore DNA synthesis was involved in this process, possibly extending a free 30 -end of the T-DNA after synthesis of the second T-DNA strand. Comparable molecules also occur in gene targeting in mammalian cells and yeast and such events were termed ectopic recombination later.

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In addition, the target locus is frequently rearranged and such events often exceed the number of precise gene targeting events (Risseeuw et al., 1995). As in double-strand break repair, deletions of parts of the target locus and substitutions with unrelated sequences were observed. The modifications were restricted to one end of the target, but the asymmetry may be caused by the experimental design that selected for precise recombination events at one end. Nevertheless, the data suggest that the ends act independently and can engage in diVerent recombination reactions. The recombination mechanisms involved in gene targeting were studied systematically by Puchta and colleagues (1996). In their system, a defective kanamycin resistance gene containing an endonuclease I-SceI recognition site served as target locus and double-strand breaks were induced at that locus by transient expression of I-SceI at the same time as the repair construct was introduced. The induction of a double-strand break stimulated homologous recombination up to 100-fold. The recombination products obtained in this reaction resulted mostly from homologous interactions at both ends of the break. However, products resulting from homologous recombination at one side and illegitimate recombination at the other side also occurred. These products are comparable to those obtained without double-strand break induction (Risseeuw et al., 1995). Repair constructs with homology to only one end of the broken molecule repair double-strand breaks with the same eYciencies as repair constructs with homology to both ends. Therefore the ends act independently and the initiation of the process occurs preferentially at only one end (Puchta, 1998a). The double-strand break model repair of Puchta et al. (1996) explains these experimental data by assuming a combination of single-strand annealing and one-sided invasion as mechanism. The double-strand break in this model is processed by an exonuclease to generate 30 single-stranded DNA ends. These ends anneal with one homologous, free end of the repair construct. This interaction results in homologous interactions at both ends if the second free 30 -end can also establish a homologous interaction with the other end. The final result is a precise replacement of target locus sequences. However, if the second end had failed to initiate a homologous interaction, this end is sealed by illegitimate recombination. Gene targeting and ectopic recombination obtained with Agrobacteriummediated transformation can also be precise processes (Reiss et al., 2000). In experiments with a seed-specific kanamycin resistance gene as target locus, gene targeting and ectopic recombination were obtained with comparable frequency. In addition, rearrangements were detected neither at the target locus nor in the replaced gene. The ectopic recombination events were characterized by DNA sequence analysis after isolation of the corresponding molecules by plasmid rescue and PCR (B. Reiss, unpublished observations). The molecules consisted of a precise partial duplication of the target locus at

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the end that had engaged in homologous recombination and a genuine T-DNA border junction at the other end. This end had integrated into repetitive DNA with no sequence homology to the sequences flanking the target locus. Such structures could originate from a T-DNA inserted at random after which one end was redirected to the target locus by a homologous interaction. After the initiation of DNA synthesis, the process was aborted at some distance from the target locus and the newly synthesized end returned to the original integration side. Such molecules are predicted from the double-strand break repair model of Puchta et al. (1996). However, because these molecules have all the hallmarks of aberrant T-DNA integration sites, they might be the result of two diVerent processes, T-DNA integration at one side and homologous recombination at the other one.

D. Plant Recombination Genes The RecA-like protein DMC1, originally isolated from lily and described as LIM15 (Kobayashi et al., 1993, 1994), is the first recombination protein identified in plants. Like the proteins from yeast and vertebrates it plays an important role in meiosis, colocalizes with RAD51 on meiotic chromosomes of lily (Terasawa et al., 1995), and is part of early recombination nodules (Anderson et al., 1997). The Arabidopsis (Sato et al., 1995; Klimyuk and Jones, 1997; Doutriaux et al., 1998) and rice (Shimazu et al., 2001) genes have also been described. In contrast to yeast and animals, expression of the Arabidopsis gene is not restricted to meiosis but is also detected in mitotically active cells (Klimyuk and Jones, 1997; Doutriaux et al., 1998). The gene is essential for male and female meiosis, and fertility is severely aVected in mutants. However, although the pattern of chromosome segregation is severely disturbed and in contrast to mouse and yeast, meiosis can be completed (Couteau et al., 1999). Another gene with a function in plant meiosis is the Arabidopsis TAM gene. This gene may have a role in coupling cell cycle progression to cell division in male meiosis (Magnard et al., 2001). The yeast SKP1 gene is a component of the ubiquitin-ligase complex SCF and is required for entry into S phase, for completion of the M phase, and plays a role in the separation of homologues in meiosis. The Arabidopsis homologue SKP1-LIKE1 is required in male meiosis and may be involved in the degradation of a protein required for homologue association (Yang et al., 1999). The AtSKP1 gene is highly expressed in all meristematic and dividing cells (Porat et al., 1998) and is part of a family with nine members in Arabidopsis (Gray et al., 1999). The SYN1 gene (Bai et al., 1999) is also required for proper completion of meiosis. syn1 mutants are male and female sterile and show defects in chromosome condensation and pairing in meiosis. SYN1 is a homologue of the Schizosaccharomyces pombe RAD21 gene and encodes a

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cohesin required for chromosome condensation and sister chromatid cohesion. SYN1 is part of a small gene family in Arabidopsis (Dong et al., 2001). DYAD, a gene necessary for progression of female meiosis, encodes an unknown protein that has a function in meiotic chromosome organization (Agashe et al., 2002). RAD51, a member of the family of RecA-like proteins, is the best analyzed recombination gene and plant homologues from tomato (Stassen et al., 1997), Arabidopsis (Doutriaux et al., 1998; K. Smith, unpublished observations), maize (Franklin et al., 1999), and Physcomitrella patens (MarkmannMulisch et al., 2002) have been described. The Arabidopsis gene is cell cycle regulated and expression peaks in S-phase. In addition, expression can be induced by gamma irradiation (Klimyuk and Jones, 1997; K. Smith, unpublished observations; Doutriaux et al., 1998). As in most eukaryotic organisms, Arabidopsis RAD51 is a single copy gene. In contrast, maize and Physcomitrella patens have two highly homologous RAD51 genes. The duplicated RAD51 genes are likely to reflect genome duplications. Maize is an ancient allotetraploid (Doebley et al., 1990) and duplicated genes, originating from the genome fusion, are common in this organism. The maize genes are redundant in function since transposon insertions in individual genes cause no phenotype. Both genes are expressed at low levels in somatic tissue, but expression is increased significantly in tissues undergoing meiosis. The proteins participate in the search for homology in chromosome pairing in maize (Franklin et al., 1999). As in maize, the two Physcomitrella RAD51 genes are actively transcribed (Markmann-Mulisch et al., 2002). In contrast to the RAD51 genes of other higher eukaryotes, both Physcomitrella genes are intron-less. This suggests the presence of an unusual recombination apparatus in this organism. The Physcomitrella genes are not entirely redundant in function, but might fulfill diVerent, but overlapping functions. Like the proteins from yeast and humans, both Physcomitrella RAD51 proteins (Ayora et al., 2002) are true DNA strand exchange proteins, interact with each other, but have a slightly diVerent mode of action. In addition, although neither of the genes complements a yeast rad51 deletion mutant, one of them, but not the other, confers a dominant negative recombination phenotype to wild-type yeast. Yeast and vertebrates have a number of additional RAD51-like genes (Shinohara and Ogawa, 1999; Thacker, 1999). RAD51-like genes are also present in Arabidopsis and, like the proteins found in other organisms, the corresponding proteins form complexes with each other (Osakabe et al., 2002). A double-strand break in yeast is processed by a complex consisting of RAD50, MRE11, and XRS2. Two of the corresponding genes have been characterized in Arabidopsis. The RAD50 gene is a single copy gene in Arabidopsis, and the protein is highly homologous to other RAD50 proteins

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(Gallego et al., 2001). The gene is transcribed as a single transcript and expressed in all tissues, but expression is strongly enhanced in flowers and other tissues containing many dividing cells. RAD50 plays a role in doublestrand break repair and meiosis since homozygous rad50 mutants are sterile and hypersensitive to DNA damage. However, in contrast to mammalian cells, the gene is not inducible by DNA damaging agents and the loss of RAD50 function is not lethal in Arabidopsis (Gallego et al., 2001). In addition, RAD50 is involved in the maintenance of telomeres and mutant cells rapidly enter a crisis with the majority of cells dying with symptoms of telomere shortening (Gallego and White, 2001). Like other RAD50 proteins, the Arabidopsis protein interacts with MRE11 (Daoudal-Cotterell et al., 2002). Also in contrast to vertebrate cells, Arabidopsis mre11 mutants are viable, but are sensitive to DNA-damaging agents, show lengthening of telomeres, and display developmental phenotypes (Bundock and Hooykaas, 2002). The SPO11 gene is involved in the initiation of meiotic recombination in yeast. In contrast to yeast, Arabidopsis has at least three SPO11 homologues (Hartung and Puchta, 2000; Grelon et al., 2001; Hartung and Puchta, 2001). The three genes are expressed in all tissues, albeit at diVerent levels. The AtSPO11-1 and AtSPO11-2 genes are diVerentially spliced, like the genes from other organisms. The AtSPO11-1 gene is the closest SPO11 homologue while AtSPO11-2 may have acquired a diVerent function since the amino acid sequence diverges at critical positions (Hartung and Puchta, 2000). Arabidopsis plants defective in AtSPO11-1 are viable, but strongly reduced in fertility. The defect aVects both male and female meiosis and is caused by a drastic reduction of bivalent formation and absence of synapsis followed by random segregation of chromosomes (Grelon et al., 2001). The function of AtSPO11-1 in plant meiosis is more like that in fungi than in animals since SPO11 is necessary for chromosome synapsis in yeast and Coprinus cinereus, but not in Drosophila and Caenorhabditis. SPO11 is a subunit A homologue of topoisomerase 6, which consists of two subunits, A and B, in archaebacteria. Although the subunit A, or SPO11, homologues are frequent in eukaryotes, the B subunit homologues were exclusively found in archaebacteria until AtTOP6B was identified in Arabidopsis (Hartung and Puchta, 2000). The AtTOP6B subunit interacts with AtSPO11-2 and AtSPO11-3, but not with AtSPO11-1, suggesting that plants, in contrast to other eukaryotes, have an archaebacterial type of topoisomerase. This gene is involved in developmental processes in Arabidopsis that require endoreduplication and defects cause severe growth abnormalities (Hartung et al., 2002; Sugimoto-Shirasu et al., 2002). RecQ proteins are DNA helicases involved in DNA replication, DNA recombination, and gene silencing. These proteins have received some attention since mutations in members of this family, in humans the Werner

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proteins, cause severe genetic disorders. The Arabidopsis genome contains six genes with homology to RecQ, but only five of them are true homologues and have both the helicase and exonuclease domains typical of these proteins (Hartung et al., 2000). RecQ homologues are also common in other plants since homologues are also present in EST collections of other plants. The KU70 and KU80 proteins play an important role in nonhomologous end joining. Like KU70 and KU80 from other organisms, the corresponding homologues of Arabidopsis (Bundock et al., 2002; Riha et al., 2002; Tamura et al., 2002; West et al., 2002) form a heterodimer with double-strand DNAbinding and single-strand DNA-dependent ATPase and helicase activity (Tamura et al., 2002; West et al., 2002). The genes are expressed at a low level in all tissues and expression is inducible by DNA damaging agents. KU70-deficient plants show no obvious growth defects. However, seedlings are hypersensitive to gamma irradiation and DNA damaging agents, but, consistent with a more important role in nonhomologous end joining, sensitivity is lost during development. In addition, ku70 mutants show a defect in the control of telomere length (Bundock et al., 2002; Riha et al., 2002). ku80 mutants are also hypersensitive to DNA-damaging agents, consistent with the role of this gene in DNA damage repair (West et al., 2002). DNA ligase IV is involved in the final step of the nonhomologous endjoining process in yeast and humans. A homologue of this gene is also present in Arabidopsis. Like the human counterparts, Arabidopsis DNA ligase IV interacts with Arabidopsis XRCC4 and has ATP-dependent ligase activity. Consistent with its role in double-strand break repair, the Arabidopsis gene is induced by gamma irradiation, but not by UV-B (West et al., 2000). In addition to DNA ligase IV, a DNA ligase I homologue with functional (Taylor et al., 1998) and biochemical (Wu et al., 2001) properties of an active ligase exists in Arabidopsis. DNA damage repair (Britt, 1995, 1996, 1999) has a direct link to recombination and therefore a selection of genes with relevance for this process is described here. Complementation studies in yeast (Fidantsef et al., 2000) and sequence analysis of the genes (Gallego et al., 2000; Liu et al., 2000) demonstrated that the Arabidopsis UVH1 gene is a homologue of the yeast RAD1 gene. The yeast RAD1 gene encodes one of the two subunits of a structurespecific endonuclease, which is involved in both UV-induced DNA damage repair and in recombination. The Arabidopsis gene is involved in the removal of nonhomologous tails and therefore has a direct role in homologous recombination in plants (Dubest et al., 2002). The Arabidopsis gene is diVerentially spliced (Vonarx et al., 2002) and expressed in all tissues, but expression is strongest in flowers (Liu et al., 2000), even in those protected from UV light (Gallego et al., 2000). Plants defective in AtRAD1 expression are hypersensitive to UV light, DNA-damaging agents (Gallego et al., 2000),

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and ionizing radiation (Dubest et al., 2002). Other nucleotide excision repair genes known from plants are homologues of RAD2 (Liu et al., 2001), RAD25 (Ribeiro et al., 1998; Costa et al., 2001), and RAD23 (Schultz and Quatrano, 1997), described from Arabidopsis and rice, respectively. DNA damage repair genes also participate in DNA integration and therefore mutants aVected in such genes may be deficient in transformation. Such mutations were described as causing deficiencies in T-DNA integration (Sonti et al., 1995). However, only the rad5 mutant was generally resistant to transformation and blocked in a step in transformation prior to T-DNA integration (Nam et al., 1998) while the uvh1 mutant was generally accessible to transformation by T-DNA (Nam et al., 1998; Preuss et al., 1999). The mismatch repair genes known from Arabidopsis include several MutS homologues (Culligan and Hays, 1997; Ade et al., 1999) and one MutL homologue, AtMLH1 (Jean et al., 1999). The MutS homologue AtMSH2 is a verified, functional mismatch repair protein (Ade et al., 2001). Mismatch repair in eukaryotes is more complex than in bacteria. The system consists of protein families, the members of which act as heterodimers with diVerent, but overlapping specificity. The mismatch repair system of plants is remarkably similar to that of other eukaryotes. Arabidopsis has the same set of MutS homologues (MSH2, MSH3, and MSH6) as other eukaryotes, but has two additional MSH6-like proteins. Also, the capacity of MutS proteins to form protein heterodimers with diVerent specificity for mismatched DNA is well conserved (Culligan and Hays, 2000). The mismatch repair genes MSH2, MSH3, and MSH6 exist also in rye and wheat (Korzun et al., 1999). Chromatin plays an important role in the regulation of gene expression and is also involved in recombination. The Arabidopsis mim mutant is sensitive to mitomycin C, UV, and gamma irradiation and shows a 3.9-fold reduction of intrachromosomal recombination. The MIM gene encodes a structural maintenance of chromosomes (SMC) protein and therefore SMC proteins in plants, as in other eukaryotes, participate both in homologous recombination and DNA damage repair. In contrast to other eukaryotes, mim mutants are viable, suggesting that the MIM protein has acquired a specialized function in plants (Mengiste et al., 1999). The MIM gene is required, but not essential, for intrachromosomal recombination since the mutation did not interfere with induction of intrachromosomal recombination by DNA damage, but overexpression of MIM stimulated the same process (Hanin et al., 2000). In a biochemical approach to study plant recombination activities, Tissier et al. (1995) purified a protein with DNA binding, renaturation, and strandexchange activities from broccoli. Unfortunately, however, the identity of this protein remained undetermined.

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E. Regulation of Recombination in Plants The Arabidopsis mutants uvs66, mkp1, and ars27A are hypersensitive to DNA-damaging agents. However, these mutants are aVected in the regulation of recombination or in signalling pathways, not in the recombination process itself. The uvs66 mutant is hypersensitive to UV irradiation and DNA-damaging agents, but also to salt stress and ABA. Nucleotide excision repair and homologous recombination are normal in this mutant and the defect aVects a signaling pathway with a link to genotoxic stress responses and ABA/salinity signaling (Albinsky et al., 1999). A defect in the AtMKP1 gene encoding a mitogen-activated protein kinase phosphatase leads to hypersensitivity to DNA damage, suggesting that a MAP kinase cascade plays a role in genotoxic stress signaling (Ulm et al., 2001). The ars27A mutant has no apparent phenotype under regular growth conditions, but DNA damage causes a tumor-like growth. The mutant is defective in one of the three S27 ribosomal protein genes. Because the mutant is unable to rapidly degrade transcripts after UV treatment, this protein could have a yet unidentified role in the elimination of damaged mRNA after UV irradiation (Revenkova et al., 1999). Poly(ADP-ribose) polymerase (PARP) is involved in DNA damage signaling to downstream acceptors and in the recovery after DNA damage. The Arabidopsis genome contains two PARP genes, AtPARP-1 and AtPARP-2, which have a similar role in plants. However, the regulation of these plant genes is diVerent from animals since both genes are rapidly and massively induced by ionizing radiation while the homologues in animals are regulated posttranscriptionally (Doucet-Chabeaud et al., 2001). The proteins associate with chromatin through cell cycle progression and are involved in chromosome dynamics (Babiychuk et al., 2001).

F. Recombination and Responses to the Environment in Plants A variety of environmental factors influence intrachromosomal recombination (Fritsch et al., 2000; I. Kovalchuk et al., 2001). Intrachromosomal recombination assays with whole plants demonstrated that nuclear pollution released by the Chernobyl accident leads to a significant increase in somatic recombination frequencies. Both the levels directly at the Chernobyl site (I. Kovalchuk et al., 1998) and the low doses found in inhabited areas around the site had a significant eVect on homologous recombination frequencies (O. Kovalchuk et al., 1999). The frequencies correlated with the levels of chromosomal aberrations observed in onion cells exposed to the same radiation (O. Kovalchuk et al., 1998). The frequencies of intrachromosomal homologous recombination were aVected by a variety of parameters, including

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the composition of soil, but responded to both acute and chronic exposure in a monotonic, dose-dependent manner. However, chronic internal exposure caused higher recombination frequencies than acute irradiation (O. Kovalchuk et al., 2000a). Crop plants react in a similar fashion to radioactive radiation since the contamination at the Chernobyl site also increased the mutation rate in the germ-line of wheat (O. Kovalchuk et al., 2000b). Analysis of somatic mutations at the whole plant level using a system monitoring the reversion frequencies of a set of defined nonsense mutations showed that the frequencies in plants exceed the estimates for other eukaryotes by at least 100-fold (I. Kovalchuk et al., 2000). The frequencies were dependent on the position of the mutation within the transgene, the location of the transgene in the genome, and the transcription rate. DNA-damaging factors like UV light, gamma rays, and MMS also induced the mutation frequency. A variety of heavy metal ions, often present as contamination in soil and water, also increase recombination and mutation frequencies in an uptake-dependent manner (O. Kovalchuk et al., 2001). Other environmental factors like UV-B irradiation also increased the frequency of homologous recombination in Arabidopsis and tobacco. The stimulatory eVect was dependent on the dose, the degree of damage, and photosynthetically active radiation. Therefore recombination may be dependent on energy supply from photosynthesis (Ries et al., 2000a). A defect in photoreactivation repair greatly stimulated recombination. Moreover, UV-B irradiation stimulated the photoreactivation and the homologous recombination pathways together, suggesting that homologous recombination is directly involved in the repair of UV-induced DNA damage in plants (Ries et al., 2000b). Biotic stress also stimulates recombination in plants. Plants attacked by the fungal pathogen Peronospora parasitica show enhanced levels of homologous recombination and this eVect is mimicked by chemical induction of plant-defense mechanisms (Lucht et al., 2002). Reactive oxygen species are generated as part of the defense reactions against pathogens. These may induce double-strand breaks and thus lead to a stimulation of homologous recombination. However, the stimulatory eVect of biotic stress may also be the result of a more general response since plants appear to generally respond to stress with an increase in homologous recombination.

G. Stimulation of Homologous Recombination and Gene Targeting 1. Induction by DNA Damage Treatments with mutagens like DNA-damaging chemicals or irradiation generally result in an induction of homologous recombination and also plants respond to such treatments the same way. A variety of artificially created or

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naturally occurring genotoxic stresses, including gamma radiation, mitomycin C, heat stress, UV light, heavy metals, and pathogen attack, induce homologous recombination in plant systems (Tovar and Lichtenstein, 1992; Lebel et al., 1993; Puchta et al., 1995; Fritsch et al., 2000; Ries et al., 2000a,b; I. Kovalchuk et al., 1998, 2001; O. Kovalchuk et al., 1999, 2000a,b, 2001; Lucht et al., 2002). In addition, the insertion of transposon leads to a dramatic stimulation of recombination when the element excises from the locus (Dooner and Martinez-Ferez, 1997a; Shalev and Levy, 1997; Xiao et al., 2000; Xiao and Peterson, 2000). However, neither transposon excision nor physical or chemical treatments have yet been used to improve gene targeting in plants. In contrast, the induction of a double-strand break at the target locus has been analyzed and results in a dramatic stimulation of gene targeting (Puchta et al., 1996; Puchta, 1998a; Reiss et al., 2000) and other homologous recombination processes such as intrachromosomal (Chiurazzi et al., 1996) and ectopic recombination (Puchta, 1999). The double-strand break is a key intermediate generated early in recombination and the stimulatory eVect suggests that initiation of the reaction at the target locus is a limiting step in gene targeting. Therefore, this approach would be a good candidate to improve gene targeting. Unfortunately, however, doublestrand breaks can be induced only at engineered recognition sites at present. This restriction prevents the general use of this technology and future work is necessary to analyze other ways to introduce a double-strand break, e.g., the use of less specific enzymes, an approach successfully used in other organisms, or targeting of DNA cleaving chemicals to the target gene. Nevertheless, the technology has potential for application and is already used to remove undesired sequences (Siebert and Puchta, 2002), e.g., selectable marker genes, from transgenic plants (Hohn et al., 2001). 2. Plant Mutants Affected in Recombination A variety of screens resulted in recombination mutants in plants, but gene targeting was not analyzed in any of them, although some of them showed elevated levels of homologous recombination. Several mutants hypersensitive to gamma rays and UV light were obtained after EMS mutagenesis (Davies et al., 1994; Jiang et al., 1997; Masson et al., 1997; Masson and Paszkowski, 1997), but most of them were not analyzed in detail and the corresponding genes have not been identified. Therefore their potential to improve gene targeting is unclear. Another set are the hypersensitive mutants described in the previous section. With the exception of mim (Mengiste et al., 1999), these were signal transduction or regulatory mutants with no change in homologous recombination and therefore not directly useful to improve gene targeting (Albinsky et al., 1999; Revenkova et al., 1999; Ulm et al., 2001). Although MIM overexpression stimulated intrachromosomal recombination

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2-fold and therefore it is involved in homologous recombination in plants (Hanin et al., 2000), its eVect on gene targeting has not yet been analyzed. The hyrec mutant resulted from a direct screen for mutants with elevated levels of recombination in tobacco (Gorbunova et al., 2000). Homologous recombination between sulfur gene alleles is stimulated 1000-fold in the dominant hyrec mutant. In addition, resistance to gamma irradiation is increased and extrachromosomal recombination is stimulated 5- to 10-fold. However, there was no eVect on intrachromosomal recombination. The dramatic stimulation of homologous recombination between chromosomes, but not of intrachromosomal recombination suggests that the HYREC gene is directly or indirectly involved in the repression of recombination between chromosomes. Such a mechanism might be a major bottleneck for gene targeting and a further analysis of this mutant would be a step ahead in understanding processes that limit gene targeting. But the HYREC gene is not tagged and other methods to isolate mutant genes in tobacco are not readily available (Gorbunova et al., 2000). Unfortunately, an analysis of gene targeting in this mutant is still missing. 3. Optimization of the Targeting Experiment and Negative Selection Early gene targeting experiments in plants (OVringa et al., 1992; Morton and Hooykaas, 1995; Vergunst and Hooykaas, 1999) used assay systems with clearly suboptimal homology between target locus and repair construct. The systems at that time used homologies of 0.4 kb (Paszkowski et al., 1988), 0.9 kb (Hrouda and Paszkowski, 1994), 1.1 kb (Halfter et al., 1992), 1.9 kb (Lee et al., 1990), and 3.6 kb (OVringa et al., 1990, 1993). These homologies are well below the value (approximately 10 kb) found to be necessary for eYcient gene targeting in mouse cells (Muller, 1999; Vasquez et al., 2001), and the extremely low frequencies of gene targeting in plants were initially attributed to this fact. Therefore new test systems with larger regions of homology were designed and those reached up to 22 kb (Puchta et al., 1996; Thykjaer et al., 1997; Puchta, 1998a; Gallego et al., 1999; Reiss et al., 2000). However, in contrast to mammalian cells, which show a strong dependence on the length of homology, the gene targeting eYciencies in plants did not increase significantly. Therefore other factors instead of or in addition to length or quality of homology may limit gene targeting in plants. However, because these studies are based on a very low number of events and include the results from diVerent target loci, plant species, and transformation methods, this conclusion has to be treated with caution. Negative selection was a powerful approach in mammalian systems to enrich for gene targeting events. The problem with this strategy in plants was the lack of a powerful negative selection system. This changed with the availability of the bacterial CodA gene as a cell autonomous negative

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selection marker for plants that could be used for selection in tissue culture (Schlaman and Hooykaas, 1997; Thykjaer et al., 1997). However, although negative selection in plants allowed a more than 1000-fold suppression of random integration (Risseeuw et al., 1997; Thykjaer et al., 1997; Gallego et al., 1999; Wang et al., 2001), this approach was not successful in plants. An exception may be rice in which a diVerent negative marker has been used (Terada et al., 2002). The basic frequencies of gene targeting in most plants may be too low to apply this technology. The transformation method has a fundamental influence on the eYciency of gene targeting in mammalian cells (Vasquez et al., 2001). However, no reports addressing this topic in plants are available. 4. Strategies Relying on Mismatch Repair Triplex forming and chimeric oligonucleotides are becoming important tools for the modification of genes and gene expression in animals. Triplex-forming oligonucleotides, like antisense or RNAi, interfere with RNA stability or gene transcription. The main field of application is transient modification of gene expression. However, triplex-forming and chimeric oligonucleotides were also used to introduce targeted modifications in animals as an alternative to gene targeting (Vasquez and Wilson, 1998; Giovannangeli and Helene, 2000; Vasquez et al., 2001). Chimeric oligonucleotides are modified RNA/DNA oligonucleotides with a hairpin structure that are rather stable and highly mutagenic molecules. They are designed to introduce single point mutations into complementary genomic target DNA and presumably cause mutations using the mismatch repair system. This technology also works in plants (Baszczynski et al., 1999; Hohn and Puchta, 1999) and mutations were introduced into two genes, a herbicide resistance gene and the jellyfish green fluorescent protein, in tobacco (Beetham et al., 1999) and maize (Zhu et al., 1999, 2000). However, the eYciencies were 3 orders of magnitude lower than those reported from animal cells (Zhu et al., 1999). In total, about one in 104 cells receiving an oligonucleotide was modified, an eYciency that is comparable to gene targeting in plants and restricts chimeric oligonucleotide technology to genes that confer a readily visible or easily selectable phenotype. An additional problem is the fidelity of the targeted modification since a substantial proportion of the modifications was not as predicted from the oligonucleotide sequence, but was either diVerent from the mutator sequence or occurred at a diVerent position. Chimeric oligonucleotide technology would have its strength in the introduction of clearly defined and small changes into the genome, combined with the advantage to avoid selectable marker genes or additional DNA sequences. These features would make it an interesting alternative to gene targeting, if the eYciencies could be improved.

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5. Manipulation of Recombination Pathways Interference with expression of genes operating in the homologous or nonhomologous pathways of recombination has a dramatic eVect on the use of these pathways. Overexpression of one single component of the homologous pathway, the RecA protein or a nuclear-targeted variant, nt-RecA, is suYcient to stimulate homologous recombination in transgenic tobacco. The plants were considerably more resistant to DNA-damaging agents, the frequency of intrachromosomal recombination was stimulated 20-fold, the fidelity of double-strand break repair improved 3.2-fold, and the levels of sister-chromatid exchange increased 2.4-fold (Reiss et al., 1996, 1997, 2000). The stimulation of homologous recombination in plants by a prokaryotic protein is unexpected. Although the RecA protein is a close homologue of the eukaryotic RAD51 protein (both proteins have a similar structure and mediate comparable biochemical reactions), they operate in the context of a set of quite diVerent environments. In addition, RAD51 requires additional proteins for full activity and it may have acquired species-specific functions. Therefore, a direct interaction of RecA with other eukaryotic recombination proteins is rather unlikely and the stimulatory eVect more likely results directly from RecA’s homology search and strand-exchange activity. This activity alone seems to suYce to drive recombination intermediates toward homologous recombination and to shift the equilibrium between the nonhomologous and homologous pathways. This is possible only if intermediates generated in the recombination process are available for diVerent activities operating in this process. Therefore this result suggests that eukaryotic recombination is not an ordered process, a conclusion also strongly supported by the diversity of recombination mechanisms that operate in eukaryotes. Another prokaryotic recombination protein, RuvC, also stimulates homologous recombination in plants (Shalev et al., 1999). RuvC mediates the step following RecA action in the homologous recombination pathway of E. coli, resolution of the Holiday structure. Like RecA, RuvC stimulates a variety of homologous recombination processes. Extrachromosomal recombination is increased 56-fold and interchromosomal recombination 12-fold. Intrachromosomal recombination, the only process tested with both, was stimulated to a degree similar (11-fold) to RecA. Stimulation of homologous processes by both a strand-exchange protein (RecA) and a resolvase (RuvC) suggests that both activities might be limiting in the homologous pathway of recombination in plants. Inhibition of the nonhomologous recombination pathway can also lead to stimulation of homologous recombination. The RAD50 protein is part of a complex that directly interacts with components of the homologous and nonhomologous pathways, an interaction that plays a major role in the

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decision as to which pathway is used. This interaction is apparently more important for the nonhomologous branch since rad50 mutants in yeast are severely deficient in nonhomologous recombination and show a weak hyperrecombination phenotype. The RAD50 gene in Arabidopsis seems to follow the same rule since its disruption stimulates intrachromosomal recombination about 10-fold (Gherbi et al., 2001). The role of RAD50 in animals is unknown since rad50 mutations are lethal in those organisms. Stimulation of homologous recombination by inactivation of major components of nonhomologous recombination would be an attractive approach to improve gene targeting; however, experimental data are lacking. 6. Links to Gene Targeting It is generally unknown how the DNA in gene targeting integrates into the genome, but it is assumed that artificially introduced DNA acts like chromosomal DNA. In this, random integration results from nonhomologous recombination while gene targeting is promoted by homologous recombination. As a consequence, stimulation of homologous or suppression of nonhomologous recombination should directly result in higher gene targeting frequencies. However, as discussed previously, gene targeting in yeast and vertebrates does not necessarily follow this rule and the same seems to apply for plants. Although a variety of homologous recombination processes are stimulated by RecA overexpression, the eYciency of gene targeting is not (Reiss et al., 1996, 2000). This eVect is independent of the target locus and also applies to gene targeting after the induction of doublestrand breaks. Because these experiments used Agrobacterium-mediated transformation, it remains possible that the single-strand DNA binding protein VirE2 that covers the transferred DNA prevented RecA from accessing its substrate. Nevertheless, the result clearly demonstrates that the eYciency of gene targeting is not exclusively governed by the presence of recombination pathways. Other factors, like the DNA delivery system, features that determine the quality of the transformed DNA as recombination substrate, or regulatory features of recombination pathways, could be as important. The result also raises the question whether the common recombination assay systems are good models for gene targeting. Intra-, inter-, and extrachromosomal recombination and resistance to DNA damage, the assays used to analyze homologous recombination in plants, may simply not detect the parameters critical for gene targeting. Unfortunately, we do not know what features would improve these systems. In addition, RecA is still the only case for which data for both homologous recombination and gene targeting are available. Therefore we have to wait for the results from other approaches before the conclusion can be generalized.

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7. Lessons from Vertebrate Systems The relationship between recombination and gene targeting has been well studied in animals (Sonoda et al., 2001; Vasquez et al., 2001). Gene targeting in mammalian cells is as problematic as in plants and random integration is by far the more common process. However, in contrast to plants, strategies such as positive–negative selection and marker target selection to suppress random integration improved the eYciencies considerably. In addition, optimization of the experimental conditions, especially the method used for transformation and the length of homology between repair construct and target locus, led to substantial improvements. Nevertheless, high gene targeting frequencies are still restricted to some few cell lines, especially the chicken DT40 and mouse ES cell lines, suggesting that the cellular system is the important factor. The expression of a variety of recombination genes in vertebrates was altered to manipulate recombination and gene targeting. Overexpression of genes acting in the homologous pathway generally stimulated homologous recombination. RAD51 overexpression stimulates homologous recombination and gene targeting, but the eVect is rather low. Other genes with a role in homologous (RAD51B, XRCC2, XRCC3, RAD54, BLM, a homologue of the RecQ helicase, BRCA-1, a tumor supressor gene encoding a protein that interacts with RAD51, RAD50, and MRE11) and nonhomologous recombination and regulatory pathways were knocked out. In most cases, the eVect on gene targeting and homologous recombination was as expected from their role in recombination and both processes were aVected in a similar fashion. Surprisingly, however, RAD52 with its central role in the homologous pathway in yeast is of relatively little importance in vertebrate cells. Altogether, the data are still incomplete and do not suggest a clear concept that would allow an improvement of gene targeting. Therefore, the gene targeting problem in animals is nearly as far away from a solution as in plants.

8. After All, What Are the Problems? In a look back one wonders what the problems with gene targeting in plants actually are. The gene targeting eYciencies in the literature range from 106 to 103. A closer look at the low eYciencies shows that they are an average of a majority of experiments with no gene replacements and some with a frequency of around 103 (Paszkowski et al., 1988; Halfter et al., 1992; Risseeuw et al., 1995; B. Reiss, unpublished observations). Gene targeting frequencies of one in a thousand are suYcient to apply this technology as a routine tool and such frequencies were suYcient in mammalian cells. Therefore, the real problems with plants must be diVerent. One is the readiness of the scientific community. Gene targeting is a rather labor-intensive tool and

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needs a considerable investment before it is ready for application. This attitude may change once the disadvantages of alternative methods became suYciently clear and the eYciencies have been reasonably improved. The other one certainly is the reproducibility of the method. As discussed, diVerences in the eYciencies between diVerent experiments are probably related to an inherent variability in gene targeting experiments. This variability could be caused by subtle and yet unrecognized diVerences in the experimental conditions that have a significant influence on eYciency. Considering the tight regulation of recombination processes in vertebrates, the cell cycle would be a good candidate and current approaches to improve gene targeting in plants address this question.

IV. Alternative Systems A. Emerging Model Plant Physcomitrella patens and Gene Targeting Gene targeting in the moss Physcomitrella patens has been detected by chance and boosted the revival of this model system (Reski et al., 1994; Cove et al., 1997; Reski, 1998, 1999; Cove, 2000; Wood et al., 2000; Schaefer, 2001; Schaefer and Zryd, 2001; Holtorf et al., 2002). Kammerer and Cove (1996) observed that retransformation of Physcomitrella transformants resulted in higher transformation frequencies, a finding readily explained with the discovery of the high eYciency of gene targeting in this organism (Schaefer and Zryd, 1997). Transformation experiments with DNA containing genomic sequences from Physcomitrella showed that the transformation frequency increased 10-fold. Moreover, the corresponding genomic loci were modified with a frequency of up to 90%. The system has been used meanwhile to knock a variety of genes out. These include the FTZ gene (13% eYciency; Strepp et al., 1998), a multiubiquitin chain-binding subunit gene, MCB1 (4% eYciency; Girod et al., 1999), a delta-6-acyl group desaturase (95% eYciency; Girke et al., 1998), a member of the CAB multigene family (33% eYciency; Hofmann et al., 1999), an adenosine 50 phosphosulfate reductase gene (42% eYciency; Koprivova et al., 2002), and five Physcomitrella expansin genes (0, 10, 80, 80, and 100% eYciencies; Schipper et al., 2002). More unpublished experiments are reviewed by Schaefer (2001). In addition, entire genomic DNA libraries were transformed back into Physcomitrella by gene targeting (Nishiyama et al., 2000; Hiwatashi et al., 2001). All of these experiments confirmed the outstanding eYciency of gene targeting obtained in Physcomitrella, although the eYciencies are somewhat variable and do not necessarily approach those of the first report (Schaefer and Zryd, 1997). These variations are minor considering the

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diVerent homologies between target locus and repair construct and the fact that diVerent loci were targeted. Nevertheless, the frequency of gene targeting in Physcomitrella is not comparable to the eYciency of yeast, as initially assumed (Schaefer, 2001), but rather to those of chicken DT40 B or mouse ES cells, especially if the requirement for homology is considered. But these eYciencies are by far suYcient to use gene targeting as a routine tool in Physcomitrella and to allow systematic functional genomics studies with this organism. However, the system is not without problems. Transformation of Physcomitrella is not as eYcient as it first seems. Transformation yields stable and unstable transformants (Schaefer, 2001). Unstable transformants remain resistant only if constant selection pressure is applied and the transgenes are lost as soon as selection is discontinued. The transgenes in this type of transformants consist of extrachromosomally replicated DNA and are presumably made up of concatamers of the transformed DNA. This class is predominant and 95% of the transformants may be unstable. Therefore, the actual yield of stable transformants is rather low. Moreover, stable transformants perish in the excess of unstable ones and have to be eliminated in a laborious and time-consuming process before stable transformants can be accessed. An additional problem is the complexity of the modified loci. In the majority of cases, a large number of copies of the targeting vector (dozens to hundreds) has integrated at the target locus (Schaefer et al., 1991; Schaefer and Zryd, 1997). Therefore clean, single copy integrations are rare and rather diYcult to find, a fact that makes sophisticated applications of gene targeting problematic in Physcomitrella. A further problem is the rather limited resources in this organism. Before a gene can be analyzed in Physcomitrella, it has to be isolated. This task can be problematic since not many Physcomitrella sequences are available. Physcomitrella genes are not well conserved to higher plant genes, especially not to the established model plant Arabidopsis. This makes sequence-based approaches, like PCR with degenerate primers or screening of libraries with heterologous probes, diYcult. However, the situation has improved considerably lately and more than 170,000 ESTs are available now in public and private databases (Rensing et al., 2002). But more sequences and information about gene and genome structure would make the work with Physcomitrella noticeably easier. Another problem, not recognized in the past, is gene redundancy. Physcomitrella is haploid in the vegetative phase. This was considered a major advantage of this system since only one allele has to be knocked out in this case to reveal a gene function. However, this organism has two highly homologous RAD51 genes and duplicated RAD51 genes are a hallmark of duplicated or fused genomes (Markmann-Mulisch et al., 2002). In addition, three more duplicated, highly homologous genes have been found shortly afterward in Physcomitrella (Imaizumi et al., 2002; Kabeya et al., 2002;

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Richter et al., 2002). These genes are single copy genes in other plants. As far as is known, the duplicated genes are at least partially redundant and both copies need be inactivated to reveal a clean phenotype (Imaizumi et al., 2002; Kabeya et al., 2002; U. Markmann-Mulisch and B. Reiss, unpublished observations). Therefore the Physcomitrella genome is likely to be duplicated and this duplication was caused by a recent event. As a consequence, there is a considerable risk that extensive gene redundancies exist in Physcomitrella and that these may mask phenotypes of knocked out genes. Studies on gene function in this organism should take this fact into account. The reasons for high gene targeting eYciencies in Physcomitrella are actively debated. In one view, the eYciency of gene targeting is correlated with the dominance of the haplophase because many primitive or haploid organisms share this feature (Schaefer and Zryd, 1997). However, lower and haploid plants are not generally highly eYcient in gene targeting and the green alga Chlamydomonas shows similar gene targeting frequencies and the same rearrangements at the target locus as are observed in higher plants (Smart and Selman, 1991; Sodeinde and Kindle, 1993; Gumpel et al., 1994). In addition, mouse ES or chicken DT40 cells are highly eYcient in gene targeting and both are diploid higher eukaryotic cells. Moreover, gene targeting in those cell lines is considerably more eYcient than in their somatic counterparts. In another view, cell cycle arrest in G2 is thought to be important since Physcomitrella protoplasts used for transformation are arrested in this stage (Reski, 1998), but this assumption remains to be proven. Moreover, taking other gene targeting eYcient cells, yeast, ES, and DT40 cells into consideration, there is nothing in common that could be a feature causing high gene targeting eYciencies. Therefore, further work is needed to reveal the biological basis for high gene targeting eYciencies.

B. Gene Targeting in Chloroplasts Plant cells have more genomes than the nuclear and one of them is the chloroplast genome. This genome has many prokaryotic features. The ribosomes are as in prokaryotes, polycistronic messages are common, and also the recombination apparatus looks prokaryotic. Homologous integration is the default pathway for DNA transformation. Chloroplasts can be transformed only if homology to the chloroplast genome is present, first shown by chloroplast transformation (Boynton et al., 1988) in the unicellular alga Chlamydomonas (Rochaix, 1995). This principle was confirmed soon after afterward in tobacco (Svab et al., 1990; Takahashi et al., 1991; Staub and Maliga, 1992; Golds et al., 1993; O’Neill et al., 1993; Svab and Maliga, 1993; Kanevski and Maliga, 1994; Zoubenko et al., 1994) and gene targeting in chloroplasts in a variety of higher plants follows the same principle

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(Maliga, 1993; Dix and Kavanagh, 1995; Rochaix, 1992, 1997; Sikdar et al., 1998; Heifetz, 2000). What distinguishes the chloroplast from the nuclear recombination machinery? In contrast to the nucleus, homologous recombination in the chloroplast is naturally highly active. Parts of the chloroplast genome, a large inverted repeat region, and other regions, recombine constantly and give rise to a population of diVerent genomes (Fejes et al., 1990). This feature is reflected in applications to generate marker-free transgenic chloroplasts (Carrer and Maliga, 1995) or to eliminate marker genes by homologous recombination (Fischer et al., 1996). I-SceI-induced double-strand breaks are highly recombinogenic in chloroplasts and are repaired by homologous recombination (Durrenberger et al., 1996). In addition, the multiple recombination events that are involved in targeted gene replacements reflect a high activity of homologous recombination (Kavanagh et al., 1999). These data confirm the presence of a highly eVective homologous recombination apparatus in chloroplasts. Moreover, the mismatch repair system in higher plant chloroplasts seems to be fairly inactive and homologous sequences are integrated with the same eYciency as nonhomologous sequences (Kavanagh et al., 1999). Interestingly, as observed with Physcomitrella, the chloroplast maintains transformed DNA as extrachromosomally replicating molecules with a high eYciency (Staub and Maliga, 1994). The recombination genes operating in the chloroplast also have apparent prokaryotic features. A close homologue of the E. coli RecA gene is present in Pisum sativum and Arabidopsis chloroplasts (Cerutti et al., 1992; Binet et al., 1993). The gene is nuclear encoded (Cao et al., 1997) and induced by DNA damage (Cerutti et al., 1993). The protein is transported to the chloroplast (Cerutti et al., 1992) and has RecA activity (Cao et al., 1997). Supporting the close relationship, dominant negative mutants of the E. coli RecA protein interfere with recombination and DNA damage repair in Chlamydomonas chloroplasts and overexpression of intact RecA protein enhanced plastid recombination, but had no eVect on DNA repair (Cerutti et al., 1995). Another Arabidopsis gene with RecA-like activities is structurally not related to RecA. This gene was detected by complementation of E. coli mutants defective in recombination and DNA damage repair. It is a nuclear gene of unknown function and encodes a chloroplast-targeted Arabidopsis protein (Pang et al., 1992). The gene is also present in a variety of other plant species. In addition, the chloroplast seems to have several resolvases (Pang et al., 1993a). The corresponding genes are also nuclear genes and encode chloroplast-targeted proteins. The genes complement E. coli mutants defective in RuvC and RecG, but the proteins have no apparent homology to these E. coli proteins. Additional, nuclear-localized genes encoding chloroplasttargeted proteins of unknown function and with no homology to E. coli proteins are involved in UV damage repair (Pang et al., 1993b).

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C. Alternatives to Gene Targeting The potential to introduce precise and well-defined modifications into the genome is restricted to gene targeting. However, other technologies also allow the production of loss of function mutants. Large populations of T-DNA and transposon-tagged populations were generated and strategies developed that allow the identification of an insertion in the gene of interest in a large number of individuals. Pooling, systematic end-sequencing of insertions, hybridization of DNA extracted from ordered plant transformant pools, and PCR-based strategies (Koes et al., 1995) now make the identification of a knockout line fairly easy (Forsthoefel et al., 1992; Azpiroz-Leehan and Feldmann, 1997; Parinov and Sundaresan, 2000; Pereira, 2000; Bouche and Bouchez, 2001). Moreover, large populations of transposon or T-DNA tagged plants exist in a variety of diVerent laboratories now (McKinney et al., 1995; Winkler et al., 1998; Krysan et al., 1999; Parinov and Sundaresan, 2000; Weigel et al., 2000; Budziszewski et al., 2001; McElver et al., 2001; Ogarkova et al., 2001; Steiner-Lange et al., 2001; Samson et al., 2002). Both the high coverage obtained with the populations and the ease of identification made screening of insertion lines extremely popular and successful. Therefore, screening of insertion libraries is an alternative to gene targeting. However, many insertions are outside of genes. Insertions in introns or 50 and 30 untranslated regions may not aVect the function of the gene, and even insertions in exons may leave residual gene activity. Therefore many knockouts are functionally not gene knockouts. In addition, the introduction of more sophisticated mutations, like single point mutations, still requires gene targeting. Moreover, knockout populations are still fairly restricted to Arabidopsis, although other model plants (maize, rice) follow. Finally, gene targeting cannot be replaced in commercial applications to precisely engineer transgenes into genomes and in doing so avoid position eVects or stabilize gene expression.

V. Concluding Remarks This review has summarized the plant gene targeting and recombination literature from its beginning up to the end of 2002. In doing so, the gene targeting problem was seen in the general context of genetic recombination in plants and had a constant eye on recombination in bacteria, yeast, and vertebrates, systems in which much more knowledge is available. This review, like any, presents a personal view of the subject and cannot cover all areas at a depth they may deserve. The reader is referred to additional reviews here (OVringa et al., 1992; Morton and Hooykaas, 1995; Puchta and Hohn,

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1996; Gorbunova and Levy, 1999; Hohn and Puchta, 1999; Mengiste and Paszkowski, 1999; Vergunst and Hooykaas, 1999; Hohn et al., 2001; Puchta, 1998b, 2000, 2002). Gene targeting in higher plants was and still is a problematic subject. The review may leave a pessimistic impression, but the gene targeting problem is not restricted to plants. Some mammalian cells are at least as diYcult and even mouse ES cells are not without problems. In contrast to plants, however, labor and time-intensive investments pay for the benefits of targeted gene replacements. Last but not least, there is a high eYciency gene targeting system in plants, the moss Physcomitrella patens, and this system is ready for gene targeting now.

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