About the Project

Meiosis is the specialized cell division used by sexually reproducing organisms to halve the chromosome complement in the gametes. During meiosis, a single round of DNA replication is followed by two successive divisions: the first (reductional) division segregates homologous chromosomes, and the second (equational) division separates sister chromatids from each other, resulting in four nuclei with a single complement of chromosomes. With the invention and application of new molecular, genetic and cytological tools in recent years, considerable advances have been made in the understanding of meiosis and in particular, in the context of this application, in understanding of the role of homologous recombination.

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division (Hamant et al., 2006). These events are initiated by induction of programmed double-strand breaks by the SPO11 protein and repair of such breaks by homologous recombination which requires many proteins, including the strand exchange activity provided by the RAD51-like protein(s). The importance of recombination in meiosis is clearly established in many organisms and evidenced by the sterility of most mutants deficient in recombination. Moreover, lethality of vertebrates’ mutants in many of these genes makes plants as multicellular eukaryotic model particularly attractive.

In the model plant Arabidopsis, major insights into the different steps of meiotic recombination have been gained by the cytological analysis of mutants impaired in meiosis. Meiotic recombination is initiated by DSB induction. In Arabidopsis mutants deficient in the Spo11-1 or Spo11-2 proteins, formation of chiasmata and bivalents is severely reduced and synapsis are absent (Grelon et al., 2001; Hartung et al., 2007b; Stacey et al., 2006). However, meiotic division proceeds resulting in ‘polyads’ with random content of DNA, instead of tetrad formation. In the subsequent step, RAD51 (the homologue of the bacterial RecA recombinase) and its meiotic paralogue DMC1 assemble on the ssDNA to form a nucleoprotein filament. This structure is essential for searches of homologous sequences and finally triggers strand invasion to form a junction between parental chromosomes, a pre-requisite for chromosomal synapsis. Consistent with this, Arabidopsis mutants of the DMC1 (Couteau et al., 1999) and RAD51 (Li et al., 2004) genes are severely affected in HR. Their chromosomes clearly fail to synapse and to form bivalents, but unlike in yeast, chromosome fragmentation caused by persisting meiotic DSBs is observed in rad51 but not dmc1 mutants. This suggests a requirement of DMC1 for the selective invasion of the homologous chromosome rather than for the repair of the SPO11-induced DSBs. These could be repaired by RAD51-mediated HR between sister chromatids or by an alternative pathway. The function of Arabidopsis RAD51 and DMC1 was suggested to be dependent on their interaction with the two paralogs of BRCA2. The absence of synapsis and the chromosome fragmentation phenotype in RNAi-brca2 plants is reminiscent of that of the double-mutant dmc1/RNAi-rad51 (Dray et al., 2006; Li et al., 2004; Siaud et al., 2004). Biochemical studies show that the RAD51 paralogs RAD51C and XRCC3 play an important role in vertebrate recombination (Liu et al., 2004) and in agreement with these findings, Atxrcc3 mutant plants reveal severe meiotic defects and a sterile phenotype (Bleuyard and White, 2004). In contrast to the rad51 and brca2 mutants, xrcc3 appears to play a post-synapsis role. As meiosis proceeds chromosome bridges and fragmentation are observed suggesting a distinct role of XRCC3 in a later phase of meiotic HR (mHR). The recent observation of Rad51C at chiasmata in mouse pachytene/diplotene supports such late role in branch migration and resolution of Holliday junctions (HJ) (Liu et al., 2007). Notwithstanding the similarity between the Rad51 paralogs and the fact that each plays important role in recombination, only the xrcc3 and rad51c mutants are sterile (Bleuyard et al., 2005). These similar proteins thus play distinct roles in the meiotic recombination process. Some eukaryotic homologs of the bacterial mismatch repair proteins MUTS and MUTL are thought to promote the crossover, branch migration and the resolution of the HJs in mHR. A mutation in the Arabidopsis MSH4 gene leads to a reduction of chiasmata number and to delayed and incomplete synapsis resulting in univalents (Higgins et al., 2004). This supports a role of Msh4 in mHR, whereby it probably acts in a heterodimeric complex with Msh5 as a sliding clamp, keeping together the homologous chromosomes engaged in HJs. More recently a nuclease involved in the resolution of meiotic crossovers could be identified in Arabidopsis: Atmus81 mutants have a moderate decrease in meiotic recombination. Indirect evidence indicates that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive (Berchowitz et al., 2007; Higgins et al., 2008). Thus, in recent years more and more key players in the different steps of meiotic recombination in plants have been defined, although no application of this knowledge for improving plant breeding has been reported.

The situation is different for gene targeting where many approaches have been undertaken to establish GT as routine in plants. DNA integration into a given chromosomal sequence, via homologous recombination is a powerful technique in prokaryotes, unicellular eukaryotes, and some multicellular eukaryotes including the moss Physcomitrella (Schaefer and Zryd, 1997), Drosophila (Rong and Golic, 2000), chicken (Bezzubova et al., 1997) and mouse (Capecchi, 1989). GT enables targeted gene disruption and replacement of endogenous loci by modified or different genes. Despite the outstanding importance of GT, frequencies are still very low in the majority of multicellular eukaryotes and the underlying reasons for this are poorly understood. Notably, the ability to modify the genome in a precise manner is underdeveloped in plants where low frequencies prevented GT to become a routine technique (Hanin et al., 2001) and little real progress has been made in the application of GT since the first successful GT report in plants (Paszkowski et al., 1988).The reports of successful GT events in plants are scarce because the experiments are still labor-intensive and non-homologous integration events outnumber by 3-5 orders of magnitude the precise, homologous integration events [for review see (Puchta, 2002)]. Only recently three new (in plants) approaches have been taken that lead to an increase in the efficiency of targeting.

The use of a positive-negative selection vector that distinguishes between homologous versus non-homologous integration has been shown to be helpful for GT at any locus in rice (Hohn and Puchta, 2003; Terada et al., 2007; Terada et al., 2002). Nevertheless the GT frequencies are still quite low (in 10-3-10-4 range), so the approach is still laborious (Hohn and Puchta, 2003).

Several previous attempts have been made to enhance gene targeting by the (over) expression of factors involved in homologous recombination. Neither expression of the bacterial RecA gene (Reiss et al., 2000), the E. coli resolvase RuvC gene (Shalev et al., 1999) nor the over-expression of the plant chromatin dynamics-related gene MIM (Hanin et al., 2000) resulted in significantly enhanced gene targeting efficiencies. However that this concept can be successfully applied was demonstrated recently by the expression of the yeast RAD54 in Arabidopsis, which produced an enhancement of targeting of more than one order of magnitude (Shaked et al., 2005). This result has been regarded as an important step in the set up of a feasible gene targeting technique in plants (Puchta and Hohn, 2005; Tzfira and White, 2005). On the other sideAn alternative approach showed that site-specfic DSB can be used to stimulate integration of a homologous sequence at a target locus by up to several orders of magnitude (Puchta et al., 1996). However, induction of DSBs in this work was achieved by expression of I-SceI, a site-specific endonuclease and this approach was thus limited to the targeting of a previously inserted I-SceI recognition site. The development of synthetic zinc-finger nucleases (ZFNs) to generate DSBs at specific genomic sites (Porteus and Carroll, 2005) was the prerequisite for applying the technique for GT in plants. A first report (Wright et al., 2005) has demonstrated that indeed GT could be strongly enhanced by this technique in tobacco cells. However, the use of ZFNs still seems to have as undesirable side effects, especially cytotoxicity caused by cleavage at off-target sites (Szczepek et al., 2007). Therefore the technique has to be improved before it is generally applicable for GT in plants.

 

 

 

References:

 

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