The dual nature of homologous recombination in plants

Review TRENDS in Genetics Vol.21 No.3 March 2005The dual nature of homologousrecombination in plantsDavid Schuermann, Jean Molinier*, Olivier Fritsch and Barbara HohnFriedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, SwitzerlandHomologous recombination creates covalent linkagesbetween DNA in regions of highly similar or identicalsequence. Recent results from several laboratories,many of them based on forward and reverse geneticsin Arabidopsis, give insights into the mechanisms of theenzymatic machinery and the involvement of chromatinin somatic and meiotic DNA recombination. Also,signaling pathways and interconnections betweenrepair pathways are being discovered. In addition,recent work shows that biotic and abiotic influencesfrom the environment can dramatically affect plantgenomes. The resulting changes in the DNA sequence,exerted at the level of somatic or meiotic tissue, mightcontribute to evolution.Finally, many specific mutation screens can be used toidentify genes of interest, including screens for radiationsensitivity and for altered levels of HR (see below).In this review, we will concentrate on newer findings inHR in meiotic and in somatic tissue. A section is devoted toinfluences of the environment on the regulation of the HRfrequency, with special reference to the possible effects ofpathogens on the stability of the plant genome. Becausenot all aspects of HR can be covered in this short article,we refer the interested reader to a previous review onHR in plants [2], to general reviews on mechanisms ofHR [3,4], to a general description of the uses ofArabidopsis as a DNA repair model [5] and to accountsof gene targeting [6,7] (Box 1).IntroductionHomologous recombination (HR) is one of the fundamentalprocesses of life. It has a dual function through its activitiesboth in meiosis and in somatic cells. It creates new linkagesin the genetic material in meiosis and in many organismsit is even required for fertility. Indeed, HR substantiallycontributes to evolution. HR is also important for repairingdamaged DNA in somatic tissue. At the same time HRbetween repeated genes must be tightly controlled to avoidunwanted gene rearrangements.There are several important reasons for studying HRand other repair processes in plants: one of them is the factthat unlike in animal systems, functional depletion ofseveral repair genes is not lethal, thus enabling the studyof their replacement activities (see below). Another lies inthe fact that the germline is created late in development.Therefore, genomic changes in somatic tissue have acertain probability of being transmitted to the next generation.This special aspect of plant development enablesnatural selection to act on new genetic traits in somatic oreven in gametophytic tissue.The recent development of genetic and genomic toolsfor Arabidopsis thaliana is one of the reasons whyanalysis of HR and other repair processes in this planthas gained an enormous boost. The completion ofArabidopsis genome sequencing in 2000 [1] is the basisfor gene discovery and through the use of T-DNA andtransposon insertions, multiple publicly available geneknockout libraries of this plant have been produced.Corresponding author: Hohn, B. (barbara.hohn@fmi.ch).* Present address: CNRS UMR6547, BIOMOVE, 24 Avenue des Landais, F-63177Aubière, France.Available online 19 January 2005Meiotic recombinationHR maintains the integrity of the genome in somatic cells;conversely, in meiotic cells it creates genetic variability byreciprocal crossovers and DNA exchanges. This results inBox 1. Homologous recombination and plant breedingGenetic improvement of crop plants, executed for many thousandsof years, depends on HR. The classical breeding procedure involvescrossing of chosen parental lines and subsequently selecting theoffspring for the desired trait. An understanding of the mechanism ofHR and its control might help to achieve this more quickly. One couldimagine that the use of hyper-recombination mutants like thosedescribed in Table 2 and mutants directly affecting meiotic recombinationcould increase the frequency of mHR. This could beexploited in breeding programs, because the time needed forcrossing and backcrossing would be considerably reduced.Resistance to stresses, such as plant pathogens, drought andsalinity, frequently has to be introduced into inbred lines. Apart fromclassical breeding, which is limited to the same or closely relatedspecies at best, transgenesis-aided breeding could be the option forthe future in many cases. Already this is common practice for theintroduction of genes from more distantly related organisms. Theability to produce targeted changes in plant genes would have atremendous impact on fundamental research and on molecularbreeding. However, the frequency of one targeted event per 10 4 –10 6transformants is too low to be routinely used [62,63] (discussed inRefs [6,7,64]). Stimulation of HR in the cells used in transgenesisprotocols might increase the chance of obtaining targeted integrationevents. Stimulation of HR might be achieved by usingradiation or chemical agents or genetically by employing mutants,as discussed above. The only reported attempt to increase thetargeting frequency using the bacterial recombinase RecA has failed[65]. The use of two counter-selectable markers flanking thetargeting gene is a valuable approach because it yielded cleantargeted events only [62]. The combination of this approach withgenetic upregulation of HR in the required tissue could lead toimproved gene replacement frequencies.www.sciencedirect.com 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.01.002

174Review TRENDS in Genetics Vol.21 No.3 March 2005Table 1. Homologous recombination genes in Arabidopsis, yeast and mammals aArabidopsis Refs Proposed function b Budding yeast MammalsATM [29] Double-strand break (DSB) signaling, cell-cycleTel1ATMcheckpointATR [72] DSB signaling, cell-cycle checkpoint Mec1 ATRBRCA2 c [22] Strand invasion – BRCA2BRU [35] Chromatin state, silencing ? ?Centrin2 [46] Nucleotide excision repair (NER) ? Centrin2DMC1 [20] Meiotic interstrand invasion Dmc1 DMC1? DSB repair? – DNA-PKERCC1 [73] NER Rad10 ERCC1INO80 [47] DSB repair, homologous recombination (HR) Ino80 INO80MIM [34,74] DNA repair and recombination Smc6? SMC6?MLH1 [26] Promotion of crossovers in meiosis Mlh1 MLH1MRE11 [17] DSB end processing Mre11 MRE11MSH4 [27] Promotion and stabilization of meiotic HR Msh4 MSH4? DNA damage signaling? – p53? DSB end processing? Xrs2 NBS1RAD1 [75–78] NER, nonhomologous overhang end removal Rad1 XPFRAD9 [39] Cell-cycle checkpoint Rad9 RAD9RAD17 [39] Cell-cycle checkpoint Rad17 RAD17RAD25 (also known as [79] NER Rad25 XPBXPB1)RAD50 [18,19] DSB end processing Rad50 RAD50RAD51 [21] Strand invasion Rad51 RAD51RAD51C [23] Holliday junction (HJ) resolution? – RAD51C? HR? Rad52 RAD52RAD54L? HR, perhaps through chromatin events? Rad54 RAD54SNM1 [44] Interstrand crosslink repair, recombination Snm1? SNM1?SPO11-1 [14,16] Induction of meiotic DSB Spo11 SPO11SPO11-2 [14,15] ? – –SPO11-3 [15] ? – –XRCC3 [22] HJ resolution? Rad57 XRCC3a The main factors involved in or potentially affecting somatic and meiotic HR are listed in alphabetical order with their function. Known homologs in budding yeast andmammals are shown. Yeast or mammalian genes for which no obvious homologs exist in Arabidopsis are highlighted in bold.b Refers to function in Arabidopsis.c Two homologs in Arabidopsis.the archaebacterial topoisomerase VI complex, whichinduces transient DSBs to disentangle DNA. In contrastto most other organisms, Arabidopsis possesses threeparalogs of Spo11 and a homolog of the topoisomerase VIBsubunit (TOP6B) [14,15] (Table 1). In an Arabidopsismutant of the SPO11-1 gene the formation of chiasmataand bivalents was severely reduced, and synapsis couldnot be observed [14,16]. Meiotic division proceeded in thespo11-1 background, resulting in ‘polyads’ of random DNAcontent instead of the typical tetrads. It remains to beelucidated whether production of residual DSBs and thereduced formation of chiasmata are due to alternativepathways or a result of the function of the two otherSPO11 paralogs. However, both of them but not SPO11-1were shown to interact with the homologs of the B subunit,suggesting a eukaryotic topoisomerase-like function [15].The budding yeast Mre11–Rad50–Nbs1 complex isassumed to participate in the Spo11-dependent inductionof DSBs and in the processing of the ends generating3 0 -protruding single stranded DNA (ssDNA). Mutantsof the Arabidopsis orthologs of Mre11 [17] and Rad50[18,19] were isolated and analyzed for their meioticphenotypes. Both mutants exhibited chromosomal fragmentationand failure of synapsis, supporting a rolefor the Arabidopsis MRE11–RAD50 complex in earlysteps of meiotic HR, presumably in processing the DNAends. In contrast to the situation in budding yeast, theArabidopsis protein complex is not required for inductionof the SPO11-mediated DSBs, because the chromosomefragmentation and the complete sterility of mre11 could bepartially suppressed in a spo11-1 background [17].In the subsequent step of yeast mHR, Rad51 (the homologof the bacterial RecA recombinase) and its meiotic paralogDmc1 assemble on the ssDNA to form a nucleoproteincomplex. This complex searches for homologous sequencesand finally triggers strand invasion to form a jointmolecule between parental chromosomes, a prerequisitefor synapsis. Consistent with this, Arabidopsis mutants inthe DMC1 [20] and RAD51 [21] genes were severelyaffected in mHR. Their chromosomes clearly failed tosynapse and to form bivalents, but unlike in yeast,chromosome fragmentation caused by persisting meioticDSBs was observed only in rad51 mutants. This suggestsa function of DMC1 in the selective invasion of thehomologous chromosome rather than in the repair ofthe SPO11-induced DSBs. These could be repaired byRAD51-mediated HR between sister chromatids or by analternative pathway. Interestingly, a DMC1 homolog couldnot be identified in Drosophila melanogaster nor inCaenorhabditis elegans, in both of which the synaptonemalcomplex is formed independently of SPO11-inducedDSBs [11]. Recently, the function of the two Arabidopsisrecombinases RAD51 and DMC1 was suggested to bedependent on their genetic and molecular interaction withthe two paralogs of BRCA2. The absence of synapsis andthe chromosome fragmentation phenotype in RNAi-brca2plants was reminiscent of that of the double-mutantdmc1/RNAi-rad51 [22], and the BRCA2 paralogswww.sciencedirect.com

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

176Review TRENDS in Genetics Vol.21 No.3 March 2005(a)RBCS1 T PRBCS2 T PRBCS3 TL U CL U CRBCS1TPRBCS2TPRBCS3TL U CRBCS3/1L U CLUC+(b)Direct repeats(c)Indirect repeatsPHG U′ U′ SG U′PHU′SG U′G U′U′ S U′ SGU SGUS+U′U′G U SGUS+(d)U′ SHPG U′(e)U′ SG U′U′ SG U′U′ S G U SGUS+U′ SG U′G U′TRENDS in GeneticsFigure I. Reporter-based homologous recombination substrates. (a) A synthetic RBCSB gene cluster containing a promoter-less firefly Luciferase gene [67]. Unequalcrossover events between sister chromatids that contain the RBCSB cluster enables the expression of the Luciferase gene, which can be scored by live-imaging using asensitive CCD camera. RBCS1, RBCS2 and RBCS3 are repeats of Ribulose-Bisphosphate-Carboxylase small subunit gene; P and T indicate the endogenous promoter andterminator for gene expression. (b,c) Intrachromosomal HR reporter constructs with direct and indirect repeats. The constructs consist of two inactive fragments(GU’, U’S) of a b-glucuronidase (GUS) gene sharing an identical stretch of 618 bp (U’). Intramolecular HR events between the repeats restore a functional gene (GUSC)with different molecular products: the direct repeats (GU’-U’S) give rise to deletion of the sequence between the two repeats, resulting in a probably short-livednonreplicative circular molecule. HR between indirect repeats (U’G-U’S) results in inversion and conservation of the sequence separating the repeats. Black trianglesrepresent the borders of the T-DNA; P indicates the CaMV viral promoter driving GUS expression; H is the hygromycin resistance gene. (d) Reporter construct used tovisualize intermolecular HR events. The major pathway that gives rise to the restoration of the GUS gene has been found to be gene conversion (depicted), but unequalreciprocal recombination between sister chromatids or allelic chromosomes and ‘pop-out’ events were also reported [70,71]. (e) In planta detection of recombination events inreporter plants harboring a stably integrated HR construct, as in (b). Histochemical GUS staining of reporter plants visualizes cells in which the GUS gene was restored by HR.www.sciencedirect.com

Review TRENDS in Genetics Vol.21 No.3 March 2005 177Table 2. Plant mutants with altered somatic homologous recombination frequency a,bMutant c Gene (AGI Pathway Assay Repair and recombination phenotype Refsaccess number)Genetic screens for genotoxic stress sensitivityxrs4 ND ND X-ray sensitivity, genetic screen X-ray, MMS and MMC sensitivity; [30,31]decreased somatic HR and increasedmHRxrs9 ND ND X-ray sensitivity, genetic screen X-ray and MMS sensitivity; decreased [30,31]somatic HR and mHRxrs11 ND ND X-ray sensitivity, genetic screen X-ray and MMC sensitivity; defective in [30,31]X-ray-mediated somatic HR inductionHyrec d ND HR Spontaneous g-ray resistant, increased interhomologs[37]HR, intrachromosomal HRunchangedMim At5 g61460 Chromatin MMS sensitivity, genetic screen MMS, UV-C, MMC and X-ray sensitivity; [34,74]decreased somatic HRBru At3 g18730 ND MMS sensitivity, genetic screen MMS, MMC, UV-C and bleomycin [35]sensitivity; increased somatic HR, TGSreleaseGenetic screens for altered HR phenotypes using chromosomal HR substratesCentrin2 At4 g37010 NER Increased HR genetic screen UV-C sensitivity; increased somatic HR, [46]defective in repair of UV-damaged DNAino80 At3 g57300 HR Altered HR genetic screen Decreased somatic HR [47]Reverse genetics and functional genomicsrad50 At2 g31970 NHEJ Homology to known protein MMS sensitivity; sterility, telomeric [18,38,40]defect, increased somatic HRrad17 At5 g66130 Cell-cyclecheckpointHomology to known protein Bleomycin and MMC sensitivity;increased somatic HR, rapid DSB repairdefect[39]rad9 At3 g05480 Cell-cyclecheckpointHomology to known proteinBleomycin and MMC sensitivity;increased somatic HR, rapid DSB repairdefectercc1 At3 g05210 NER Homology to known protein UV-C sensitivity; ECR with nonhomologousoverhangs reduced, decreasedchromosomal HRsnm1 At3 g26680 HR? Functional genomics Bleomycin and H 2 O 2 sensitivity;impaired induction of HR by H 2 O 2 andflagellin but not bleomycin, MMC andMMSMutants previously isolated for other functionrad1 (uvh1,xpf)At5 g41150 NER Known UV-C-sensitive mutant UV-B, UV-C, g-ray and cisplatin sensitivity;ECR with nonhomologous overhangsreduced, impaired HR inductionby bleomycinuvr2 (phr1) At1 g12370 Photorepair Known UV-B-sensitive mutant UV-B sensitivity; defective in CPDsphotorepair, slightly increased somaticHRcim3 ND Plant defense Uncharacterized defense mutant Constitutively activated systemicacquired resistance, increased somaticHRvtc1 (soz1) At2 g39770 Vitamin C Known UV-B-sensitive mutant UV-B and H 2 O 2 sensitivity; ascorbicaciddeficient, increased somatic HRTt4 (chs) At5 g13930 Flavonoid Known UV-B-sensitive mutant UV-B sensitivity; chalcone synthasedeficient, increased somatic HRTt5 (chi) At3 g55120 Flavonoid Known UV-B-sensitive mutant UV-B sensitivity; chalcone isomerasedeficient, increased somatic HR[39][73][44][75–78,80][41,53,81][43][82,83], (G. Ries,PhD thesis,Basel University,1999)[82,84], (G. Ries,PhD thesis,Basel University,1999)[82,84], (G. Ries,PhD thesis,Basel University,1999)a Abbreviations: CPDs, cyclobutane pyrimidine dimers; ECR, extrachromosomal HR; HR, homologous recombination; mHR, meiotic HR; MMC, mitomycin C; MMS,methylmethane sulfonate; ND, not determined; NER, nucleotide excision repair; NHEJ, nonhomologous end-joining; TGS, transcriptional gene silencing.b Within the past few years, the use of various strategies proved to be successful and complementary in identifying a large number of mutants involved in somatic HR inplants. Such mutants are listed in the top part of the table with the expected impaired pathway and a summary of their phenotypes. These include: (i) mutants originatingfrom indirect genetic screens (i.e. by searching for mutations resulting in altered sensitivity to genotoxic stress and by subsequently testing their effect on HR); (ii) mutantsobtained through direct genetic screening of mutagenized Arabidopsis lines carrying a chromosomal HR reporter construct; (iii) mutants originating from reverse geneticapproaches (i.e. testing publicly available Arabidopsis mutants in genes homologous to recombination or repair genes known from other organisms) or genes inferred fromfunctional genomics studies; and (iv) some additional previously characterized mutants are listed in the lower part of the table. These were identified for other phenotypesand only subsequently shown to affect HR.c Alternative names are indicated in parenthesis.d Hyrec is in Nicotiana tabacum; all other mutants are in Arabidopsis thaliana.www.sciencedirect.com

178Review TRENDS in Genetics Vol.21 No.3 March 2005DSBChromatin eventsBRU ?INO80 ?MIM ?RAD50MRE11?ATMATRKU70KU80NHEJSignalingPathwaydetermination(ii) Strand invasionRAD51 ?(i) 5′- 3′ resectionRAD54L ?DSB repair modelSDSA modelInterconnectionwith other repairSNM1 pathways(vii) First strand invasionand synthesisCentrinRAD17RAD9(iii) New DNA synthesisBranch migration(viii) Second strand invasionand synthesis(iv) Holliday junctionresolutionRAD1ERCC1(ix) New strands annealingand synthesis(x) Gene conversion withunaffected template(v) No crossing-over(vi) Crossing-over offlanking markersTRENDS in GeneticsFigure 2. The DSB and synthesis-dependent strand-annealing (SDSA) repair models of HR in plants. (i–vi) DSB repair model for HR. (i) Initially the DSB is resected in 5 0 -to-3 0direction, producing 3 0 single-stranded DNA ends. (ii) The 3 0 ends invade a homologous DNA duplex forming a DNA crossover, or Holliday junction (HJ), and providingprimers to initiate new DNA synthesis. (iii) The region of heteroduplex is extended through branch migration of the HJ away from the initial crossover site. (iv) HJs areresolved by cleavage of either the crossed (green arrows) or the non-crossed (black arrows) strands of the junction. Resolution in the same orientation does not affect theflanking markers (v), whereas a mixed resolution of the two HJs does (vi). (vii–x) The SDSA model for HR. (vii) One of the 3 0 single-stranded tails invades the homologousduplex, priming DNA synthesis. (viii) The other 3 0 single-stranded tail can also subsequently invade the homologous duplex and prime synthesis. After displacement from thedonor duplex (ix) the nascent strand pairs with the other 3 0 single-stranded tail and DNA synthesis and ligation complete repair (x). Factors known to be involved in HR inplants, most of which are discussed in the main text, are depicted.Recently, the combination of various approaches toisolate mutants in somatic HR has led to a dramaticincrease in our knowledge about plant HR, its pathways,its components and its regulation (Table 2 and Figure 2).Plant mutants screened for sensitivity to UV, methylmethanesulfonate (MMS) and X-rays were tested forHR; publicly available random insertional mutants ingenes known from other organisms to be involved in HRand in genes inferred from functional genomic approacheswere analyzed. Finally, direct screens for altered levelsof HR in populations of mutagenized recombinationtester-lines were conducted. All approaches yielded HRmutants, and proved to be complementary (Table 2).Strikingly, genetic screen approaches yielded many novelgenes or genes not expected to be involved in theregulation of HR.Some of the X-ray-sensitive mutants were found to bechanged at the level of somatic recombination, some atthe level of mHR and some at both levels (xrs mutants inTable 2). The screens for MMS sensitivity yielded the twomutants mim [34] and bru [35]. The MIM gene encodes aprotein closely related to the structural maintenance ofchromosomes (SMC) family of proteins, which have acentral role in chromosome organization and dynamics inbudding yeast. The yeast MIM homolog, Smc6, hasrecently been implicated in interchromosomal and sisterchromatidrecombination [36]. In the bru mutant, inwhich the stability of heterochromatin is affected, levels ofsomatic HR are increased. It is therefore entirely possiblethat the elevated HR levels in bru plants reflect anincreased sensitivity to DNA-damaging agents or animproved accessibility to the sites of damage for repairfactors rather than a direct involvement in the repairmachinery. It is of interest to mention the only describedhyper-recombinogenic mutant isolated in a non-Arabidopsisspecies: a tobacco mutant obtained in a transposonwww.sciencedirect.com

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]. 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