The dual nature of homologous recombination in plants

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Review TRENDS in Genetics Vol.21 No.3 March 2005 179mutagenesis approach exhibited a 1000-fold increasedfrequency of mitotic recombination between homologouschromosomes, as measured by the endogenous ‘sulfur’system, while leaving intrachromosomal HR unaffected.Unfortunately, the mutation could not be characterized [37].In the first reverse genetic approach mentioned above,that is, searching for and characterizing publicly availableinsertional mutants in specific genes, Arabidopsismutants in the genes RAD50, RAD9 and RAD17 couldbe obtained that exhibited increased levels of HR, linkedwith defects in DSB repair and hypersensitivity togenotoxic stress [38,39]. RAD9 and RAD17 were shownto be involved in the cell-cycle checkpoint in both yeastand Arabidopsis and might point to a connection betweenDNA damage signaling and HR regulation (Figure 2). InArabidopsis both rad50 mutants, which are hypersensitiveto DNA damage and hyper-recombinogenic in somatictissue, and rad51 mutant plants [21] grow normally butare sterile. This is in sharp contrast to the situation inmouse where mutations in both genes abolish cell viability(as discussed in Refs [21,40]). Mammalian cells obviouslyrequire the activity of both genes for cell cycle progression.The influence of the rad50, rad9 and rad17 mutationson the level of HR seems to be indirect, which could pointto a regulated balance between repair activities andaltered availability of damaged sites to repair machineries.A similar explanation could hold for plants mutatedin the photolyase gene [41], impaired in scavenging ofreactive oxygen species [42] and in plants upregulated forpathogen defense [43]: there, increased HR could be due toincreased levels of DNA damage.The snm1 mutant was obtained by searching forinsertional mutants in genes inferred from functionalgenomic studies to be differentially regulated by genotoxicstress. It is defective in the induction of somatic HR byspecific oxidative stress (Table 2) and could unravel theexistence in plants of a specific recombinational repairpathway for oxidatively induced DNA damage [44,45].The approaches to identifying plant genes involved inHR by knocking out candidate genes will of course notyield new and unsuspected genes; only a screen directlytesting for HR will permit this. In two different screensemploying the recombination substrate lines shown inFigure Ic and Id in Box 2, a multitude of mutants changedin HR levels were identified and isolated (O. Fritsch et al.,unpublished). Also, in one of these mutants the reason forthe strongly induced frequency of HR seems to be indirect:a T-DNA-mediated knockout of the Centrin2 gene, encodinga protein involved in recognition of DNA damage thatcan be repaired by the nucleotide excision repair (NER)pathway, led to a UV-C-sensitive, hyper-recombinogenicmutant. This points to a novel connection between anearly step of NER and HR [46]. Another mutant, with areduced level of HR, was mutated in a gene coding forINO80, a member of the SWI–SNF ATPase family [47].The efficiency of NHEJ seemed unaffected in the mutant.In vitro interaction of this protein with nucleosomessuggested that INO80 acts through modification ofchromatin structure, possibly at the site of DNA repair.Interestingly, the yeast Ino80 protein, as part of theINO80 complex, has recently been shown to be recruited towww.sciencedirect.comDSBs, in a manner dependent on phosphorylated histoneH2A [48]. Further studies are necessary to find out if theArabidopsis INO80 – or the potential complex thatcontains it – is also recruited to sites of DNA damage,and if such recruitment leads to the specific engagement ofthe HR machinery.The influence of the environment on somatic HRThe in planta assays described (Box 2) enable thequantitative assessment of HR events, thus permittingthe analysis of the influence of abiotic and biotic agents onHR. The effects of gamma radiation, UV radiation, heavymetals and pathogens on the physiological behavior ofplants have been amply described. However, the impactof these environmental factors on HR in genomes of theaffected plants, discussed in more general terms byMcClintock [49], has not been thoroughly studied untilrecently. Exposure of plants to gamma radiation – eitherresulting from accidents in atomic power stations or in anexperimental setup – was expected to lead to higher levelsof DSBs. Indeed, the use of plants carrying recombinationreporter constructs yielded HR frequencies that directlyreflected the level of radioactive contamination [50].Especially interesting was the fact that even low levelsof gamma radiation could be recorded with surprisingaccuracy, opening the possibility for environmental biomonitoringregimes [51]. Other analyses of abiotic influenceson HR in Arabidopsis have included tests with UV-B[52], heavy metals [53], X-rays, heat shock and UV-C(reviewed in Ref. [2]). The mechanisms by which themeasured increases in rates of HR are established are notknown, but direct induction of DSBs, the activity ofreactive oxygen species and the upregulation of DNArepair activities are likely to contribute.The influence of biotic factors such as pathogens onplants has been studied from various aspects, but only afew studies focus on the possible effects of pathogens onthe genomes of plants. Elevated mutation rates in maizefollowing virus infection have been reported, but moleculardata are lacking [54]. Inoculation of Arabidopsis linescontaining an HR reporter gene with Peronospora parasiticaled to stimulation of somatic HR. The same effectwas observed when pathogen defense mechanisms werechemically or genetically activated in the absence of apathogen [43]. Infection of tobacco plants carryingrecombination markers with tobacco mosaic virus (TMV)yielded increased recombination rates not only in inoculatedleaves but also in noninoculated leaves [55].Treatment of plants with UV-B or with TMV led to anincreased rate of HR-mediated genetically fixed restorationof the functional reporter gene (Box 2) in theoffspring [52,55]: the number of seedlings that werecompletely b-glucuronidase- or luciferase-positive wasgreater in the offspring of treated plants than in those ofuntreated plants. It is unclear, however, whether theseinduced events were due to somatic HR events late inplant development, that is, before or after meiosis, or dueto enhanced rates of mHR.An increase in either somatic or meiotic HR couldfacilitate evolutionary adaptation of plant populationsto stressful environments. Possible substrates for
180Review TRENDS in Genetics Vol.21 No.3 March 2005recombination events in plants are the numerous diseaseresistance genes spread in clusters throughout thegenome [56]. New resistance genes can be created bysequence exchange between genes in the same or differentclusters, as was reported for the maize Rp1 complex[57,58] and the tomato Cf-4 and Cf-9 loci ([59] and Refstherein). Infection of plants with pathogens can thereforelead to an increased frequency of rearrangements betweenresistance genes and thereby possibly to an adaptiveadvantage in pathogen defense. It can thus be proposedthat abiotic and biotic environmental influences affect thegenotypes of plants on two levels: (i) the level of mutation(so far shown for HR and point mutation [60]) enabling alarge genetic diversity in the population; and (ii) the levelof selection in the offspring.Concluding remarksRecent forward and reverse genetics of Arabidopsis andother plants has permitted the isolation of numerousgenes involved in meiotic and somatic HR. Some of thesewere expected to have a role in HR based on the roles ofhomologous genes in other organisms, but in other casestheir role in plants deviates in some aspect from that oftheir homologs in animals and yeast. It is predicted thatmany more functions will be unraveled in the not toodistant future.Specific questions for researchers include: (i) are therefunctional homologs of RAD52 and other repair proteins inplants (Table 1); (ii) which other proteins are directlyinvolved in the mechanics of meiotic and somatic HR inplants and; (iii) how does HR work on the level ofchromatin? Examples for the contribution of chromatinin HR have been cited (MIM, INO80 and perhaps BRU),and additional proteins acting on chromatin at the stage offormation of SPO11-dependent DSBs can be expected forplants, as has been shown for C. elegans [61]. Finally, howis somatic HR regulated, or more specifically, how aredifferent repair activities orchestrated?Another crucial aspect for plants is the influence of theenvironment on the plant genome: ‘illustrations fromnature.support the conclusion that stress, and thegenome’s reaction to it, may underlie many formations ofnew species’ [49]. We will have to analyze how this stressis perceived, how signals are sent through cells andorganisms, and how and with what specificity changes areintroduced into the genomes. Are these changes geneticallystable, are they epigenetic in nature or are thereepigenetic intermediates?AcknowledgementsWe thank the members of our group for stimulating discussions and theNovartis Research Foundation for financial support. J.M. was funded bythe EU project PLANTREC N8 QLG2-CT-2001–01397.References1 Arabidopsis-Genome-Initiative. (2000) Analysis of the genomesequence of the flowering plant Arabidopsis thaliana. Nature 408,796–8152 Puchta, H. and Hohn, B. (1996) From centiMorgans to base pairs:homologous recombination in plants. Trends Plant Sci. 1, 340–3483 Paques, F. and Haber, J.E. (1999) Multiple pathways of recombinationinduced by double-strand breaks in Saccharomyces cerevisiae. Microbiol.Mol. Biol. Rev. 63, 349–4044 West, S.C. 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The dual nature of homologous recombination in plants

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