The dual nature of homologous recombination in plants

Review TRENDS in Genetics Vol.21 No.3 March 2005 175interacted with both DMC1 and RAD51 in a yeast-twohybridanalysis. With the same technique interactionswithin the RAD51 paralogs RAD51 and XRCC3, andbetween XRCC3 and RAD51C were detected [23]. Inagreement with these findings, xrcc3 mutant plantsrevealed severe meiotic defects and a sterility phenotype[24]. In contrast to the previously described mutants,synapsis in xrcc3 appeared to be unaffected and bivalentswere formed normally. As meiosis proceeded chromosomebridges and fragmentation were observed suggesting adistinct role of XRCC3 in a later phase of mHR; mostprobably, branch migration and resolution of Hollidayjunctions (HJ) are dependent on both XRCC3 andRAD51C [25].Some eukaryotic homologs of the bacterial mismatchrepair proteins MutS and MutL are thought to promotethe crossover, branch migration and the resolution of theHJs in mHR. An Arabidopsis mlh1 mutant was describedthat lacked an obvious meiotic or somatic phenotype [26],although the nature of this allele did not permit a finalconclusion. A mutation in the Arabidopsis MSH4 gene ledto a reduction of chiasmata number and to delayed andincomplete synapsis resulting in univalents [27]. Thissupports a role of MSH4 in mHR, whereby it probablyacts in a heterodimeric complex with MSH5 as a slidingclamp, keeping together the homologous chromosomesengaged in HJs, as was recently proposed for the humanMSH4–MSH5 complex [28].An interesting aspect of plant meiosis is the lack of acheckpoint, which controls the progression of meiosis,provoking meiotic arrest in yeast and apoptosis inanimals. All of the mutants described above proceedthrough meiosis despite containing aberrant chromosomefigures, enabling more extensive studies on meioticchromosome dynamics. Nevertheless, the Arabidopsishomolog of Ataxia Telangiectasia-Mutated (ATM), whichsignals from DSB to checkpoint and repair pathways inother eukaryotes, was shown to be involved in somaticDNA damage response and clearly has a function inmeiotic recombination; mutants in this gene reveal achromosome fragmentation phenotype similar to that ofthe xrcc3 mutant [29]. This suggests that ArabidopsisATM controls mHR at SPO11 induced DSBs, but not theintegrity of meiotic chromosomes.It is to be expected that more genes acting in mHR willbe identified with time and that careful analyses ofmultiple mutations will decipher plant meiosis. Forinstance, some of the mutants isolated in a screen forX-ray sensitivity had also changed levels of meioticrecombination [30,31], but the genes responsible for thisphenotype have not been isolated yet (Table 2).Somatic recombinationDSBs are among the most dangerous forms of DNAdamage; a single unrepaired DSB in yeast, even in adispensable gene, can result in cell death [32]. DSBs arecaused by both cell-external and internal factors: ionizingradiation is an example of an external factor, DNAreplication across a nick is an internal factor, whereasreactive oxygen species can accumulate in plant cells as aconsequence of pathogen attack and/or of intrinsicmetabolic activities. Numbers are not available for plants,but it has been estimated that 5–10% of first passageprimary fibroblasts from mice or humans have a chromosomebreak (discussed in Ref. [33]). In plants andvertebrates most of these breaks are repaired by nonhomologous-endjoining (NHEJ), an error-prone process.HR enables repair in a precise fashion. However, HR insomatic tissue is not only active in repair, but also inchanging the copy number of genes, as is already evidentin recombination products originating from HR in transgenicmarker genes (see Figure I in Box 2). Repair willhave to be fast and efficient, whereas changes in copynumber will have to be modulated in a subtle way. Therepair of DNA damage in somatic cells has to be understoodin terms of the repair pathways involved and interms of modulation of these pathways as influenced bydevelopment and the environment.Box 2. Marker genes for assays of homologous recombinationMapped phenotypic markers (such as color markers in maize kernelsor the classic pea markers used by Mendel) or molecular markers areusually employed for the analysis of mHR frequencies in plants,recombination being assayed in the recombinant progeny. To enablethe measurement and visualization of somatic and meiotic HR eventsup to the resolution of a single cell, special substrates were developed.The basis of the technique is the insertion into the plant genome(using Agrobacterium tumefaciens) of transgenes permitting selectableor screenable detection of HR events. Markers based on selection(for instance involving the creation by recombination of antibioticresistance) are more difficult to use and require cell culture of planttissue (reviewed in Ref. [2]). The most valuable constructs enable theanalysis of HR at the plant level using a visible reporter. Such a systemwas recently developed, consisting of an endogenous cluster ofRibulose-Bisphosphate-Carboxylase small subunit (RBCS) genes,one of which is fused to a promoterless Luciferase reporter gene(Figure Ia). Recombination between the RBCS repeats enables theluciferase gene to switch from an inactive state to an active screenablestate. This locus-specific integrated recombination substrate can revealmeiotic intergenic unequal crossing-over between sister chromatids[66,67]. Moreover, somatic recombination events, occurring in lateflower or very early embryo development, can also be identified.The analysis of HR in somatic plant tissue was previously restrictedto the use of ill-characterized natural systems [68,69]. The use of newmarker genes has revolutionized the research field within the past15 years. Currently used reporter-based constructs consist of tandemrepeats of a disrupted b-glucuronidase (GUS) oraLuciferase (LUC)gene (Figure Ib–d; schemes shown for GUS). Different orientations,relative to each other, of the disrupted genes enable the scoring ofintramolecular (Figure Ib,c) or intermolecular HR events (Figure Id). Inthe second case a chromosomal homolog or a sister chromatid servesas recombination partner [2,70]. Thereby, different types of recombinationprocesses could lead to a functional reporter gene: pop-out,unequal reciprocal exchange and gene conversion [70], which is –shown in Figure Id – possibly the most frequent [71]. The structure ofthe recombination substrate trap could not be designed to enable adistinction between interchromatid or interchromosomal recombination.Only the use of molecular markers flanking the repeats of thedisrupted marker gene would permit this. It should be noted that inplants containing the intramolecular constructs (Figure Ib,c) HR canalso occur between sister chromatids and homologous chromosomesalthough at a lower frequency than expected for intramolecularrecombination.www.sciencedirect.com