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Review TRENDS in Genetics Vol.21 No.3 March 2005 The dual nature of homologous recombination in plants David Schuermann, Jean Molinier*, Olivier Fritsch and Barbara Hohn Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland Homologous recombination creates covalent linkages between DNA in regions of highly similar or identical sequence. Recent results from several laboratories, many of them based on forward and reverse genetics in Arabidopsis, give insights into the mechanisms of the enzymatic machinery and the involvement of chromatin in somatic and meiotic DNA recombination. Also, signaling pathways and interconnections between repair pathways are being discovered. In addition, recent work shows that biotic and abiotic influences from the environment can dramatically affect plant genomes. The resulting changes in the DNA sequence, exerted at the level of somatic or meiotic tissue, might contribute to evolution. Finally, many specific mutation screens can be used to identify genes of interest, including screens for radiation sensitivity and for altered levels of HR (see below). In this review, we will concentrate on newer findings in HR in meiotic and in somatic tissue. A section is devoted to influences of the environment on the regulation of the HR frequency, with special reference to the possible effects of pathogens on the stability of the plant genome. Because not all aspects of HR can be covered in this short article, we refer the interested reader to a previous review on HR in plants [2], to general reviews on mechanisms of HR [3,4], to a general description of the uses of Arabidopsis as a DNA repair model [5] and to accounts of gene targeting [6,7] (Box 1). Introduction Homologous recombination (HR) is one of the fundamental processes of life. It has a dual function through its activities both in meiosis and in somatic cells. It creates new linkages in the genetic material in meiosis and in many organisms it is even required for fertility. Indeed, HR substantially contributes to evolution. HR is also important for repairing damaged DNA in somatic tissue. At the same time HR between repeated genes must be tightly controlled to avoid unwanted gene rearrangements. There are several important reasons for studying HR and other repair processes in plants: one of them is the fact that unlike in animal systems, functional depletion of several repair genes is not lethal, thus enabling the study of their replacement activities (see below). Another lies in the fact that the germline is created late in development. Therefore, genomic changes in somatic tissue have a certain probability of being transmitted to the next generation. This special aspect of plant development enables natural selection to act on new genetic traits in somatic or even in gametophytic tissue. The recent development of genetic and genomic tools for Arabidopsis thaliana is one of the reasons why analysis of HR and other repair processes in this plant has gained an enormous boost. The completion of Arabidopsis genome sequencing in 2000 [1] is the basis for gene discovery and through the use of T-DNA and transposon insertions, multiple publicly available gene knockout libraries of this plant have been produced. Meiotic recombination HR maintains the integrity of the genome in somatic cells; conversely, in meiotic cells it creates genetic variability by reciprocal crossovers and DNA exchanges. This results in Corresponding author: Hohn, B. ([email protected]). * Present address: CNRS UMR6547, BIOMOVE, 24 Avenue des Landais, F-63177 Aubière, France. Available online 19 January 2005 Box 1. Homologous recombination and plant breeding Genetic improvement of crop plants, executed for many thousands of years, depends on HR. The classical breeding procedure involves crossing of chosen parental lines and subsequently selecting the offspring for the desired trait. An understanding of the mechanism of HR and its control might help to achieve this more quickly. One could imagine that the use of hyper-recombination mutants like those described in Table 2 and mutants directly affecting meiotic recombination could increase the frequency of mHR. This could be exploited in breeding programs, because the time needed for crossing and backcrossing would be considerably reduced. Resistance to stresses, such as plant pathogens, drought and salinity, frequently has to be introduced into inbred lines. Apart from classical breeding, which is limited to the same or closely related species at best, transgenesis-aided breeding could be the option for the future in many cases. Already this is common practice for the introduction of genes from more distantly related organisms. The ability to produce targeted changes in plant genes would have a tremendous impact on fundamental research and on molecular breeding. However, the frequency of one targeted event per 104–106 transformants is too low to be routinely used [62,63] (discussed in Refs [6,7,64]). Stimulation of HR in the cells used in transgenesis protocols might increase the chance of obtaining targeted integration events. Stimulation of HR might be achieved by using radiation or chemical agents or genetically by employing mutants, as discussed above. The only reported attempt to increase the targeting frequency using the bacterial recombinase RecA has failed [65]. The use of two counter-selectable markers flanking the targeting gene is a valuable approach because it yielded clean targeted events only [62]. The combination of this approach with genetic upregulation of HR in the required tissue could lead to improved 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 Review TRENDS in Genetics Vol.21 No.3 March 2005 random combinations of alleles and traits, which are subjected to natural selection and therefore contribute to the evolution of an organism. Meiosis is a key event in the life cycle of all sexually reproducing eukaryotic organisms, initiating the transition from the diploid to the haploid phase. Differentiated cells undergo a reductional meiotic division characterized by a single round of DNA replication and chromosome condensation. In most species the induction of double-strand breaks (DSBs), the alignment of the maternal and paternal chromosomes and the recombination between homologous sequences are mechanistically linked resulting in full synapsis (see Glossary). The haploid gametes are the products of two subsequent rounds of meiotic chromosome segregation. Surprisingly, recent cytological, genetic and molecular investigations have revealed many species-specific variations in the meiotic process previously thought to be highly conserved (reviewed in Refs [8–11]). The behavior of plant mutants in different phases of meiosis – from the differentiation of the meiocytes, the arrangement of sister chromatids and chromosomes in the synaptonemal complex, to DNA recombination, the formation of bivalents and finally the separation of chromosomes – has been extensively described elsewhere [12,13]. Herein we focus on the recent findings concerning Arabidopsis gene products that participate mechanistically in the process of meiotic HR (mHR) and compare them with components that are active (a) DSB induction End-processing SPO11-2 SPO11-3 and and TOP6B TOP6B ? ? SPO11-1 Recombinase loading RAD51 paralogs? MRE11– RAD50– ? 173 Glossary Bivalent: a distinct chromosomal figure in prophase I of meiosis, consisting of the fully condensed homologous chromosomes linked by chiasmata. Chiasma (plural chiasmata): a meiotic X-shaped structure seen at the separation of the bivalents; resulting from the sequence exchange between homologous chromosomes by HR. Crossover: DNA exchange between homologous chromosomes. Gene cluster: multiple similar genes at a given locus, arising from gene duplications and recombination. Holliday junction (HJ): an intermediate four-stranded branched DNA structure in the HR process whose resolution results in crossover/noncrossover of the flanking markers (see Figures 1 and 2). Meiocyte: a differentiated cell subsequently undergoing a meiotic division. Polyad: an aberrant product of meiotic division observed in some mutants, forming an atypical number of spores with random chromosome number and DNA content. Synapsis: a complete lengthwise association of the homologous chromosomes during early prophase I of meiosis. Synaptonemal complex (SC): a tripartite proteinaceous structure, formed between aligned homologous chromosomes. Tetrad: a product of meiotic division with four equally distributed haploid sets of chromosomes. Univalent: a chromosomal figure observed in mutants that fail to synapse and to form bivalents. in mHR in the budding yeast Saccharomyces cerevisiae (Figure 1 and Table 1). Meiotic recombination is initiated by the induction of DSBs. In yeast, the endonuclease Spo11 introduces DNA DSBs in meiotic chromosomes, triggering strand exchange and crossover, which is essential for synapsis in some organisms [10]. Spo11 shows homology to the A subunit of Strand invasion RAD51C Resolvases? ATM XRCC3 ? BRCA2 Branch migration HJ resolution RAD51C RAD54-L ? MSH4– MSH5? ? XRCC3 SC (b) ? RAD51 and DMC1 ? MLH1– MLH3? RAD54-like ? Mre11– Rad50– Nbs1 Spo11 Mre11– Rad50– Nbs1 (c) Rad52 Rad55 Rad57 Msh4– Msh5 ? Rad54 Mms4– Mus81 SC Rad51 and Dmc1 (d) Mec1 Leptotene Chromosome condensation Zygotene Pairing of chromosomes Initation of SC Pre-synapsis Mlh1, Mlh3 and others Pachytene Bivalents formed Synapsis Rad54 Diplotene Chromosome separation Disassembly of SC chiasmata Diakinesis Chromosome separation and condensation Post-synapsis TRENDS in Genetics Figure 1. Mechanism and proteins involved in meiotic homologous recombination embedded in the choreography of meiosis. Important processes of HR (a) and the proteins whose functions were assigned to meiotic HR in Arabidopsis (b) and in budding yeast (c) are placed into the temporal progression of meiotic prophase I and its typical chromosomal figures (d). The involvement of proteins in meiotic HR was reported (colored and filled shapes), suggested (empty shapes) or unknown (dashed shapes). Note that sister chromatids are only shown in the first and last phases. www.sciencedirect.com 174 Review TRENDS in Genetics Vol.21 No.3 March 2005 Table 1. Homologous recombination genes in Arabidopsis, yeast and mammalsa Arabidopsis ATM Refs [29] ATR BRCA2c BRU Centrin2 DMC1 ? ERCC1 INO80 MIM MLH1 MRE11 MSH4 ? ? RAD1 RAD9 RAD17 RAD25 (also known as XPB1) RAD50 RAD51 RAD51C ? RAD54L? SNM1 SPO11-1 SPO11-2 SPO11-3 XRCC3 [72] [22] [35] [46] [20] [73] [47] [34,74] [26] [17] [27] [75–78] [39] [39] [79] [18,19] [21] [23] [44] [14,16] [14,15] [15] [22] Proposed functionb Double-strand break (DSB) signaling, cell-cycle checkpoint DSB signaling, cell-cycle checkpoint Strand invasion Chromatin state, silencing Nucleotide excision repair (NER) Meiotic interstrand invasion DSB repair? NER DSB repair, homologous recombination (HR) DNA repair and recombination Promotion of crossovers in meiosis DSB end processing Promotion and stabilization of meiotic HR DNA damage signaling? DSB end processing? NER, nonhomologous overhang end removal Cell-cycle checkpoint Cell-cycle checkpoint NER Budding yeast Tel1 Mammals ATM Mec1 – ? ? Dmc1 – Rad10 Ino80 Smc6? Mlh1 Mre11 Msh4 – Xrs2 Rad1 Rad9 Rad17 Rad25 ATR BRCA2 ? Centrin2 DMC1 DNA-PK ERCC1 INO80 SMC6? MLH1 MRE11 MSH4 p53 NBS1 XPF RAD9 RAD17 XPB DSB end processing Strand invasion Holliday junction (HJ) resolution? HR? HR, perhaps through chromatin events? Interstrand crosslink repair, recombination Induction of meiotic DSB ? ? HJ resolution? Rad50 Rad51 – Rad52 Rad54 Snm1? Spo11 – – Rad57 RAD50 RAD51 RAD51C RAD52 RAD54 SNM1? SPO11 – – XRCC3 a 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 and mammals 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, which induces transient DSBs to disentangle DNA. In contrast to most other organisms, Arabidopsis possesses three paralogs of Spo11 and a homolog of the topoisomerase VIB subunit (TOP6B) [14,15] (Table 1). In an Arabidopsis mutant of the SPO11-1 gene the formation of chiasmata and bivalents was severely reduced, and synapsis could not be observed [14,16]. Meiotic division proceeded in the spo11-1 background, resulting in ‘polyads’ of random DNA content instead of the typical tetrads. It remains to be elucidated whether production of residual DSBs and the reduced formation of chiasmata are due to alternative pathways or a result of the function of the two other SPO11 paralogs. However, both of them but not SPO11-1 were shown to interact with the homologs of the B subunit, suggesting a eukaryotic topoisomerase-like function [15]. The budding yeast Mre11–Rad50–Nbs1 complex is assumed to participate in the Spo11-dependent induction of DSBs and in the processing of the ends generating 3 0 -protruding single stranded DNA (ssDNA). Mutants of the Arabidopsis orthologs of Mre11 [17] and Rad50 [18,19] were isolated and analyzed for their meiotic phenotypes. Both mutants exhibited chromosomal fragmentation and failure of synapsis, supporting a role for the Arabidopsis MRE11–RAD50 complex in early steps of meiotic HR, presumably in processing the DNA ends. In contrast to the situation in budding yeast, the Arabidopsis protein complex is not required for induction of the SPO11-mediated DSBs, because the chromosome www.sciencedirect.com fragmentation and the complete sterility of mre11 could be partially suppressed in a spo11-1 background [17]. In the subsequent step of yeast mHR, Rad51 (the homolog of the bacterial RecA recombinase) and its meiotic paralog Dmc1 assemble on the ssDNA to form a nucleoprotein complex. This complex searches for homologous sequences and finally triggers strand invasion to form a joint molecule between parental chromosomes, a prerequisite for synapsis. Consistent with this, Arabidopsis mutants in the DMC1 [20] and RAD51 [21] genes were severely affected in mHR. Their chromosomes clearly failed to synapse and to form bivalents, but unlike in yeast, chromosome fragmentation caused by persisting meiotic DSBs was observed only in rad51 mutants. This suggests a function of DMC1 in the selective invasion of the homologous chromosome rather than in the repair of the SPO11-induced DSBs. These could be repaired by RAD51-mediated HR between sister chromatids or by an alternative pathway. Interestingly, a DMC1 homolog could not be identified in Drosophila melanogaster nor in Caenorhabditis elegans, in both of which the synaptonemal complex is formed independently of SPO11-induced DSBs [11]. Recently, the function of the two Arabidopsis recombinases RAD51 and DMC1 was suggested to be dependent on their genetic and molecular interaction with the two paralogs of BRCA2. The absence of synapsis and the chromosome fragmentation phenotype in RNAi-brca2 plants was reminiscent of that of the double-mutant dmc1/RNAi-rad51 [22], and the BRCA2 paralogs Review TRENDS in Genetics Vol.21 No.3 March 2005 interacted with both DMC1 and RAD51 in a yeast-twohybrid analysis. With the same technique interactions within the RAD51 paralogs RAD51 and XRCC3, and between XRCC3 and RAD51C were detected [23]. In agreement with these findings, xrcc3 mutant plants revealed severe meiotic defects and a sterility phenotype [24]. In contrast to the previously described mutants, synapsis in xrcc3 appeared to be unaffected and bivalents were formed normally. As meiosis proceeded chromosome bridges and fragmentation were observed suggesting a distinct role of XRCC3 in a later phase of mHR; most probably, branch migration and resolution of Holliday junctions (HJ) are dependent on both XRCC3 and RAD51C [25]. 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. An Arabidopsis mlh1 mutant was described that lacked an obvious meiotic or somatic phenotype [26], although the nature of this allele did not permit a final conclusion. A mutation in the Arabidopsis MSH4 gene led to a reduction of chiasmata number and to delayed and incomplete synapsis resulting in univalents [27]. 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, as was recently proposed for the human MSH4–MSH5 complex [28]. An interesting aspect of plant meiosis is the lack of a checkpoint, which controls the progression of meiosis, provoking meiotic arrest in yeast and apoptosis in animals. All of the mutants described above proceed through meiosis despite containing aberrant chromosome figures, enabling more extensive studies on meiotic chromosome dynamics. Nevertheless, the Arabidopsis homolog of Ataxia Telangiectasia-Mutated (ATM), which signals from DSB to checkpoint and repair pathways in other eukaryotes, was shown to be involved in somatic DNA damage response and clearly has a function in 175 meiotic recombination; mutants in this gene reveal a chromosome fragmentation phenotype similar to that of the xrcc3 mutant [29]. This suggests that Arabidopsis ATM controls mHR at SPO11 induced DSBs, but not the integrity of meiotic chromosomes. It is to be expected that more genes acting in mHR will be identified with time and that careful analyses of multiple mutations will decipher plant meiosis. For instance, some of the mutants isolated in a screen for X-ray sensitivity had also changed levels of meiotic recombination [30,31], but the genes responsible for this phenotype have not been isolated yet (Table 2). Somatic recombination DSBs are among the most dangerous forms of DNA damage; a single unrepaired DSB in yeast, even in a dispensable gene, can result in cell death [32]. DSBs are caused by both cell-external and internal factors: ionizing radiation is an example of an external factor, DNA replication across a nick is an internal factor, whereas reactive oxygen species can accumulate in plant cells as a consequence of pathogen attack and/or of intrinsic metabolic activities. Numbers are not available for plants, but it has been estimated that 5–10% of first passage primary fibroblasts from mice or humans have a chromosome break (discussed in Ref. [33]). In plants and vertebrates most of these breaks are repaired by nonhomologous-end joining (NHEJ), an error-prone process. HR enables repair in a precise fashion. However, HR in somatic tissue is not only active in repair, but also in changing the copy number of genes, as is already evident in recombination products originating from HR in transgenic marker genes (see Figure I in Box 2). Repair will have to be fast and efficient, whereas changes in copy number will have to be modulated in a subtle way. The repair of DNA damage in somatic cells has to be understood in terms of the repair pathways involved and in terms of modulation of these pathways as influenced by development and the environment. Box 2. Marker genes for assays of homologous recombination Mapped phenotypic markers (such as color markers in maize kernels or the classic pea markers used by Mendel) or molecular markers are usually employed for the analysis of mHR frequencies in plants, recombination being assayed in the recombinant progeny. To enable the measurement and visualization of somatic and meiotic HR events up 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 selectable or screenable detection of HR events. Markers based on selection (for instance involving the creation by recombination of antibiotic resistance) are more difficult to use and require cell culture of plant tissue (reviewed in Ref. [2]). The most valuable constructs enable the analysis of HR at the plant level using a visible reporter. Such a system was recently developed, consisting of an endogenous cluster of Ribulose-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 the luciferase gene to switch from an inactive state to an active screenable state. This locus-specific integrated recombination substrate can reveal meiotic intergenic unequal crossing-over between sister chromatids [66,67]. Moreover, somatic recombination events, occurring in late flower or very early embryo development, can also be identified. www.sciencedirect.com The analysis of HR in somatic plant tissue was previously restricted to the use of ill-characterized natural systems [68,69]. The use of new marker genes has revolutionized the research field within the past 15 years. Currently used reporter-based constructs consist of tandem repeats of a disrupted b-glucuronidase (GUS) or a Luciferase (LUC) gene (Figure Ib–d; schemes shown for GUS). Different orientations, relative to each other, of the disrupted genes enable the scoring of intramolecular (Figure Ib,c) or intermolecular HR events (Figure Id). In the second case a chromosomal homolog or a sister chromatid serves as recombination partner [2,70]. Thereby, different types of recombination processes 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 of the recombination substrate trap could not be designed to enable a distinction between interchromatid or interchromosomal recombination. Only the use of molecular markers flanking the repeats of the disrupted marker gene would permit this. It should be noted that in plants containing the intramolecular constructs (Figure Ib,c) HR can also occur between sister chromatids and homologous chromosomes although at a lower frequency than expected for intramolecular recombination. Review 176 TRENDS in Genetics Vol.21 No.3 March 2005 (a) RBCS1 T P RBCS2 T P RBCS3 T LUC T P RBCS2 P RBCS3 T T LUC RBCS1 RBCS3/1 LUC LUC LUC+ Direct repeats U′ U′ Indirect repeats U′ H G (c) S H G (b) P U′ S P G U′ U′ G G U′ S U′ S U S GUS+ H U′ S G U′ U S GUS+ (d) U′ G (e) U′ P U′ S G U′ U′ U′ S S G U′ G U S G U′ G U′ GUS+ U′ S TRENDS in Genetics Figure I. Reporter-based homologous recombination substrates. (a) A synthetic RBCSB gene cluster containing a promoter-less firefly Luciferase gene [67]. Unequal crossover events between sister chromatids that contain the RBCSB cluster enables the expression of the Luciferase gene, which can be scored by live-imaging using a sensitive CCD camera. RBCS1, RBCS2 and RBCS3 are repeats of Ribulose-Bisphosphate-Carboxylase small subunit gene; P and T indicate the endogenous promoter and terminator 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-lived nonreplicative circular molecule. HR between indirect repeats (U’G-U’S) results in inversion and conservation of the sequence separating the repeats. Black triangles represent 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 to visualize 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 unequal reciprocal recombination between sister chromatids or allelic chromosomes and ‘pop-out’ events were also reported [70,71]. (e) In planta detection of recombination events in reporter 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 177 Table 2. Plant mutants with altered somatic homologous recombination frequencya,b Mutantc Gene (AGI Pathway access number) Genetic screens for genotoxic stress sensitivity xrs4 ND ND Assay Repair and recombination phenotype Refs X-ray sensitivity, genetic screen [30,31] xrs9 ND ND X-ray sensitivity, genetic screen xrs11 ND ND X-ray sensitivity, genetic screen Hyrecd ND HR Spontaneous Mim At5 g61460 Chromatin MMS sensitivity, genetic screen Bru At3 g18730 ND MMS sensitivity, genetic screen X-ray, MMS and MMC sensitivity; decreased somatic HR and increased mHR X-ray and MMS sensitivity; decreased somatic HR and mHR X-ray and MMC sensitivity; defective in X-ray-mediated somatic HR induction g-ray resistant, increased interhomologs HR, intrachromosomal HR unchanged MMS, UV-C, MMC and X-ray sensitivity; decreased somatic HR MMS, MMC, UV-C and bleomycin sensitivity; increased somatic HR, TGS release Genetic screens for altered HR phenotypes using chromosomal HR substrates Centrin2 At4 g37010 NER Increased HR genetic screen ino80 At3 g57300 HR Reverse genetics and functional genomics rad50 At2 g31970 NHEJ Altered HR genetic screen Homology to known protein rad17 At5 g66130 Cell-cycle checkpoint Homology to known protein rad9 At3 g05480 Cell-cycle checkpoint Homology to known protein ercc1 At3 g05210 NER Homology to known protein snm1 At3 g26680 HR? Functional genomics [30,31] [37] [34,74] [35] UV-C sensitivity; increased somatic HR, defective in repair of UV-damaged DNA Decreased somatic HR [46] MMS sensitivity; sterility, telomeric defect, increased somatic HR Bleomycin and MMC sensitivity; increased somatic HR, rapid DSB repair defect Bleomycin and MMC sensitivity; increased somatic HR, rapid DSB repair defect UV-C sensitivity; ECR with nonhomologous overhangs reduced, decreased chromosomal HR Bleomycin and H2O2 sensitivity; impaired induction of HR by H2O2 and flagellin but not bleomycin, MMC and MMS [18,38,40] Mutants previously isolated for other function rad1 (uvh1, At5 g41150 NER xpf) Known UV-C-sensitive mutant uvr2 (phr1) At1 g12370 Photorepair Known UV-B-sensitive mutant cim3 ND Plant defense Uncharacterized defense mutant vtc1 (soz1) At2 g39770 Vitamin C Known UV-B-sensitive mutant Tt4 (chs) At5 g13930 Flavonoid Known UV-B-sensitive mutant UV-B sensitivity; chalcone synthase deficient, increased somatic HR Tt5 (chi) At3 g55120 Flavonoid Known UV-B-sensitive mutant UV-B sensitivity; chalcone isomerase deficient, increased somatic HR a [30,31] UV-B, UV-C, g-ray and cisplatin sensitivity; ECR with nonhomologous overhangs reduced, impaired HR induction by bleomycin UV-B sensitivity; defective in CPDs photorepair, slightly increased somatic HR Constitutively activated systemic acquired resistance, increased somatic HR UV-B and H2O2 sensitivity; ascorbicacid deficient, increased somatic HR [47] [39] [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) 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. 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 in plants. 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 originating from 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) mutants obtained through direct genetic screening of mutagenized Arabidopsis lines carrying a chromosomal HR reporter construct; (iii) mutants originating from reverse genetic approaches (i.e. testing publicly available Arabidopsis mutants in genes homologous to recombination or repair genes known from other organisms) or genes inferred from functional genomics studies; and (iv) some additional previously characterized mutants are listed in the lower part of the table. These were identified for other phenotypes and 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. b www.sciencedirect.com Review 178 TRENDS in Genetics Vol.21 No.3 March 2005 DSB ATM Signaling ATR Chromatin events INO80 ? BRU ? ? RAD50 KU70 KU80 NHEJ MRE11 MIM ? Interconnection with other repair SNM1 pathways (i) 5′- 3′ resection RAD51 ? Pathway determination Centrin RAD17 RAD54L ? DSB repair model RAD9 SDSA model (ii) Strand invasion (vii) First strand invasion and synthesis (iii) New DNA synthesis Branch migration (viii) Second strand invasion and synthesis RAD1 ERCC1 (iv) Holliday junction resolution (ix) New strands annealing and synthesis (x) Gene conversion with unaffected template (v) No crossing-over (vi) Crossing-over of flanking markers TRENDS in Genetics Figure 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 0 direction, 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 providing primers 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 are resolved 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 the flanking 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 homologous duplex, priming DNA synthesis. (viii) The other 3 0 single-stranded tail can also subsequently invade the homologous duplex and prime synthesis. After displacement from the donor 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 in plants, most of which are discussed in the main text, are depicted. Recently, the combination of various approaches to isolate mutants in somatic HR has led to a dramatic increase 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, methylmethane sulfonate (MMS) and X-rays were tested for HR; publicly available random insertional mutants in genes known from other organisms to be involved in HR and in genes inferred from functional genomic approaches were analyzed. Finally, direct screens for altered levels of HR in populations of mutagenized recombination tester-lines were conducted. All approaches yielded HR mutants, and proved to be complementary (Table 2). Strikingly, genetic screen approaches yielded many novel genes or genes not expected to be involved in the regulation of HR. Some of the X-ray-sensitive mutants were found to be changed at the level of somatic recombination, some at www.sciencedirect.com the level of mHR and some at both levels (xrs mutants in Table 2). The screens for MMS sensitivity yielded the two mutants mim [34] and bru [35]. The MIM gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family of proteins, which have a central role in chromosome organization and dynamics in budding yeast. The yeast MIM homolog, Smc6, has recently been implicated in interchromosomal and sisterchromatid recombination [36]. In the bru mutant, in which the stability of heterochromatin is affected, levels of somatic HR are increased. It is therefore entirely possible that the elevated HR levels in bru plants reflect an increased sensitivity to DNA-damaging agents or an improved accessibility to the sites of damage for repair factors rather than a direct involvement in the repair machinery. It is of interest to mention the only described hyper-recombinogenic mutant isolated in a non-Arabidopsis species: a tobacco mutant obtained in a transposon Review TRENDS in Genetics Vol.21 No.3 March 2005 mutagenesis approach exhibited a 1000-fold increased frequency of mitotic recombination between homologous chromosomes, 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 available insertional mutants in specific genes, Arabidopsis mutants in the genes RAD50, RAD9 and RAD17 could be obtained that exhibited increased levels of HR, linked with defects in DSB repair and hypersensitivity to genotoxic stress [38,39]. RAD9 and RAD17 were shown to be involved in the cell-cycle checkpoint in both yeast and Arabidopsis and might point to a connection between DNA damage signaling and HR regulation (Figure 2). In Arabidopsis both rad50 mutants, which are hypersensitive to DNA damage and hyper-recombinogenic in somatic tissue, and rad51 mutant plants [21] grow normally but are sterile. This is in sharp contrast to the situation in mouse where mutations in both genes abolish cell viability (as discussed in Refs [21,40]). Mammalian cells obviously require the activity of both genes for cell cycle progression. The influence of the rad50, rad9 and rad17 mutations on the level of HR seems to be indirect, which could point to a regulated balance between repair activities and altered availability of damaged sites to repair machineries. A similar explanation could hold for plants mutated in the photolyase gene [41], impaired in scavenging of reactive oxygen species [42] and in plants upregulated for pathogen defense [43]: there, increased HR could be due to increased levels of DNA damage. The snm1 mutant was obtained by searching for insertional mutants in genes inferred from functional genomic studies to be differentially regulated by genotoxic stress. It is defective in the induction of somatic HR by specific oxidative stress (Table 2) and could unravel the existence in plants of a specific recombinational repair pathway for oxidatively induced DNA damage [44,45]. The approaches to identifying plant genes involved in HR by knocking out candidate genes will of course not yield new and unsuspected genes; only a screen directly testing for HR will permit this. In two different screens employing the recombination substrate lines shown in Figure Ic and Id in Box 2, a multitude of mutants changed in HR levels were identified and isolated (O. Fritsch et al., unpublished). Also, in one of these mutants the reason for the strongly induced frequency of HR seems to be indirect: a T-DNA-mediated knockout of the Centrin2 gene, encoding a protein involved in recognition of DNA damage that can be repaired by the nucleotide excision repair (NER) pathway, led to a UV-C-sensitive, hyper-recombinogenic mutant. This points to a novel connection between an early step of NER and HR [46]. Another mutant, with a reduced level of HR, was mutated in a gene coding for INO80, 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 nucleosomes suggested that INO80 acts through modification of chromatin structure, possibly at the site of DNA repair. Interestingly, the yeast Ino80 protein, as part of the INO80 complex, has recently been shown to be recruited to www.sciencedirect.com 179 DSBs, in a manner dependent on phosphorylated histone H2A [48]. Further studies are necessary to find out if the Arabidopsis INO80 – or the potential complex that contains it – is also recruited to sites of DNA damage, and if such recruitment leads to the specific engagement of the HR machinery. The influence of the environment on somatic HR The in planta assays described (Box 2) enable the quantitative assessment of HR events, thus permitting the analysis of the influence of abiotic and biotic agents on HR. The effects of gamma radiation, UV radiation, heavy metals and pathogens on the physiological behavior of plants have been amply described. However, the impact of these environmental factors on HR in genomes of the affected plants, discussed in more general terms by McClintock [49], has not been thoroughly studied until recently. Exposure of plants to gamma radiation – either resulting from accidents in atomic power stations or in an experimental setup – was expected to lead to higher levels of DSBs. Indeed, the use of plants carrying recombination reporter constructs yielded HR frequencies that directly reflected the level of radioactive contamination [50]. Especially interesting was the fact that even low levels of gamma radiation could be recorded with surprising accuracy, opening the possibility for environmental biomonitoring regimes [51]. Other analyses of abiotic influences on 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 the measured increases in rates of HR are established are not known, but direct induction of DSBs, the activity of reactive oxygen species and the upregulation of DNA repair activities are likely to contribute. The influence of biotic factors such as pathogens on plants has been studied from various aspects, but only a few studies focus on the possible effects of pathogens on the genomes of plants. Elevated mutation rates in maize following virus infection have been reported, but molecular data are lacking [54]. Inoculation of Arabidopsis lines containing an HR reporter gene with Peronospora parasitica led to stimulation of somatic HR. The same effect was observed when pathogen defense mechanisms were chemically or genetically activated in the absence of a pathogen [43]. Infection of tobacco plants carrying recombination markers with tobacco mosaic virus (TMV) yielded increased recombination rates not only in inoculated leaves but also in noninoculated leaves [55]. Treatment of plants with UV-B or with TMV led to an increased rate of HR-mediated genetically fixed restoration of the functional reporter gene (Box 2) in the offspring [52,55]: the number of seedlings that were completely b-glucuronidase- or luciferase-positive was greater in the offspring of treated plants than in those of untreated plants. It is unclear, however, whether these induced events were due to somatic HR events late in plant development, that is, before or after meiosis, or due to enhanced rates of mHR. An increase in either somatic or meiotic HR could facilitate evolutionary adaptation of plant populations to stressful environments. Possible substrates for 180 Review TRENDS in Genetics Vol.21 No.3 March 2005 recombination events in plants are the numerous disease resistance genes spread in clusters throughout the genome [56]. New resistance genes can be created by sequence exchange between genes in the same or different clusters, as was reported for the maize Rp1 complex [57,58] and the tomato Cf-4 and Cf-9 loci ([59] and Refs therein). Infection of plants with pathogens can therefore lead to an increased frequency of rearrangements between resistance genes and thereby possibly to an adaptive advantage in pathogen defense. It can thus be proposed that abiotic and biotic environmental influences affect the genotypes of plants on two levels: (i) the level of mutation (so far shown for HR and point mutation [60]) enabling a large genetic diversity in the population; and (ii) the level of selection in the offspring. Concluding remarks Recent forward and reverse genetics of Arabidopsis and other plants has permitted the isolation of numerous genes involved in meiotic and somatic HR. Some of these were expected to have a role in HR based on the roles of homologous genes in other organisms, but in other cases their role in plants deviates in some aspect from that of their homologs in animals and yeast. It is predicted that many more functions will be unraveled in the not too distant future. Specific questions for researchers include: (i) are there functional homologs of RAD52 and other repair proteins in plants (Table 1); (ii) which other proteins are directly involved in the mechanics of meiotic and somatic HR in plants and; (iii) how does HR work on the level of chromatin? Examples for the contribution of chromatin in HR have been cited (MIM, INO80 and perhaps BRU), and additional proteins acting on chromatin at the stage of formation of SPO11-dependent DSBs can be expected for plants, as has been shown for C. elegans [61]. Finally, how is somatic HR regulated, or more specifically, how are different repair activities orchestrated? Another crucial aspect for plants is the influence of the environment on the plant genome: ‘illustrations from nature.support the conclusion that stress, and the genome’s reaction to it, may underlie many formations of new species’ [49]. We will have to analyze how this stress is perceived, how signals are sent through cells and organisms, and how and with what specificity changes are introduced into the genomes. Are these changes genetically stable, are they epigenetic in nature or are there epigenetic intermediates? Acknowledgements We thank the members of our group for stimulating discussions and the Novartis Research Foundation for financial support. J.M. was funded by the EU project PLANTREC N8 QLG2-CT-2001–01397. References 1 Arabidopsis-Genome-Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815 2 Puchta, H. and Hohn, B. (1996) From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci. 1, 340–348 www.sciencedirect.com 3 Paques, F. and Haber, J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 4 West, S.C. (2003) Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4, 435–445 5 Hays, J.B. (2002) Arabidopsis thaliana, a versatile model system for study of eukaryotic genome-maintenance functions. DNA Repair (Amst.) 1, 579–600 6 Reiss, B. (2003) Homologous recombination and gene targeting in plant cells. Int. Rev. Cytol. 228, 85–139 7 Britt, A.B. and May, G.D. (2003) Re-engineering plant gene targeting. Trends Plant Sci. 8, 90–95 8 Petronczki, M. et al. (2003) Un ménage à quatre: the molecular biology of chromosome segregation in meiosis. Cell 112, 423–440 9 Zickler, D. and Kleckner, N. (1999) Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33, 603–754 10 Shaw, P. and Moore, G. (1998) Meiosis: vive la différence! Curr. Opin. Plant Biol. 1, 458–462 11 Villeneuve, A.M. and Hillers, K.J. (2001) Whence meiosis? Cell 106, 647–650 12 Caryl, A.P. et al. (2003) Dissecting plant meiosis using Arabidopsis thaliana mutants. J. Exp. Bot. 54, 25–38 13 Bhatt, A.M. et al. (2001) Plant meiosis: the means to 1N. Trends Plant Sci. 6, 114–121 14 Hartung, F. and Puchta, H. (2000) Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana. Nucleic Acids Res. 28, 1548–1554 15 Hartung, F. and Puchta, H. (2001) Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants. Gene 271, 81–86 16 Grelon, M. et al. (2001) AtSPO11-1 is necessary for efficient meiotic recombination in plants. EMBO J. 20, 589–600 17 Puizina, J. et al. (2004) Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis. Plant Cell 16, 1968–1978 18 Gallego, M.E. et al. (2001) Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity. Plant J. 25, 31–41 19 Bleuyard, J.Y. et al. (2004) Meiotic defects in the Arabidopsis rad50 mutant point to conservation of the MRX complex function in early stages of meiotic recombination. Chromosoma 113, 197–203 20 Couteau, F. et al. (1999) Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis. Plant Cell 11, 1623–1634 21 Li, W. et al. (2004) The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis. Proc. Natl. Acad. Sci. U. S. A. 101, 10596–10601 22 Siaud, N. et al. (2004) Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1. EMBO J. 23, 1392–1401 23 Osakabe, K. et al. (2002) Molecular cloning and characterization of RAD51-like genes from Arabidopsis thaliana. Plant Mol. Biol. 50, 71–81 24 Bleuyard, J.Y. and White, C.I. (2004) The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis. EMBO J. 23, 439–449 25 Liu, Y. et al. (2004) RAD51C is required for Holliday junction processing in mammalian cells. Science 303, 243–246 26 Jean, M. et al. (1999) Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana. Mol. Gen. Genet. 262, 633–642 27 Higgins, J.D. et al. (2004) The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis. Genes Dev. 18, 2557–2570 28 Snowden, T. et al. (2004) hMSH4-hMSH5 recognizes Holliday junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes. Mol. Cell 15, 437–451 29 Garcia, V. et al. (2003) AtATM is essential for meiosis and the somatic response to DNA damage in plants. Plant Cell 15, 119–132 30 Masson, J.E. et al. (1997) Mutants of Arabidopsis thaliana hypersensitive to DNA-damaging treatments. Genetics 146, 401–407 31 Masson, J.E. and Paszkowski, J. (1997) Arabidopsis thaliana mutants altered in homologous recombination. Proc. Natl. Acad. Sci. U. S. A. 94, 11731–11735 Review TRENDS in Genetics Vol.21 No.3 March 2005 32 Bennett, C.B. et al. (1993) Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc. Natl. Acad. Sci. U. S. A. 90, 5613–5617 33 Lieber, M.R. et al. (2003) Mechanism and regulation of human nonhomologous DNA end-joining. Nat. Rev. Mol. Cell Biol. 4, 712–720 34 Mengiste, T. et al. (1999) An SMC-like protein is required for efficient homologous recombination in Arabidopsis. EMBO J. 18, 4505–4512 35 Takeda, S. et al. (2004) BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis. Genes Dev. 18, 782–793 36 Onoda, F. et al. (2004) SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae. DNA Repair (Amst.) 3, 429–439 37 Gorbunova, V. et al. (2000) A new hyperrecombinogenic mutant of Nicotiana tabacum. Plant J. 24, 601–611 38 Gherbi, H. et al. (2001) Homologous recombination in planta is stimulated in the absence of Rad50. EMBO Rep. 2, 287–291 39 Heitzeberg, F. et al. (2004) The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination. Plant J. 38, 954–968 40 Gallego, M.E. and White, C.I. (2001) RAD50 function is essential for telomere maintenance in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 98, 1711–1716 41 Ries, G. et al. (2000) UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation. Proc. Natl. Acad. Sci. U. S. A. 97, 13425–13429 42 Filkowski, J. et al. (2004) Systemic plant signal triggers genome instability. Plant J. 38, 1–11 43 Lucht, J.M. et al. (2002) Pathogen stress increases somatic recombination frequency in Arabidopsis. Nat. Genet. 30, 311–314 44 Molinier, J. et al. (2004) SNM-dependent recombinational repair of oxidatively induced DNA damage in Arabidopsis thaliana. EMBO Rep. 5, 994–999 45 Molinier, J. et al. Dynamic response of plant genome to ultraviolet radiation and other genotoxic stresses. Mutat. Res. (in press) 46 Molinier, J. et al. (2004) CENTRIN2 modulates homologous recombination and nucleotide excision repair in Arabidopsis. Plant Cell 16, 1633–1643 47 Fritsch, O. et al. (2004) The INO80 protein controls homologous recombination in Arabidopsis thaliana. Mol. Cell 16, 479–485 48 van Attikum, H. et al. (2004) Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodelling with DNA double-strand break repair. Cell 119, 777–788 49 McClintock, B. (1984) The significance of responses of the genome to challenge. Science 226, 792–801 50 Kovalchuk, I. et al. (1998) Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nat. Biotechnol. 16, 1054–1059 51 Kovalchuk, O. et al. (1999) Radiation hazard caused by the Chernobyl accident in inhabited areas of Ukraine can be monitored by transgenic plants. Mutat. Res. 446, 49–55 52 Ries, G. et al. (2000) Elevated UV-B radiation reduces genome stability in plants. Nature 406, 98–101 53 Kovalchuk, O. et al. (2001) A sensitive transgenic plant system to detect toxic inorganic compounds in the environment. Nat. Biotechnol. 19, 568–572 54 Brakke, M.K. (1984) Mutations, the aberrant ratio phenomenon, and virus infection of maize. Annu. Rev. Phytopathol. 22, 77–94 55 Kovalchuk, I. et al. (2003) Pathogen-induced systemic plant signal triggers DNA rearrangements. Nature 423, 760–762 56 Leister, D. (2004) Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene. Trends Genet. 20, 116–122 57 Ramakrishna, W. et al. (2002) Structural analysis of the maize rp1 complex reveals numerous sites and unexpected mechanisms of local rearrangement. Plant Cell 14, 3213–3223 58 Richter, T.E. et al. (1995) New rust resistance specificities associated with recombination in the Rp1 complex in maize. Genetics 141, 373–381 www.sciencedirect.com 181 59 Wulff, B.B. et al. (2004) Genetic variation at the tomato Cf-4/Cf-9 locus induced by EMS mutagenesis and intralocus recombination. Genetics 167, 459–470 60 Kovalchuk, I. et al. (2000) Genome-wide variation of the somatic mutation frequency in transgenic plants. EMBO J. 19, 4431–4438 61 Reddy, K.C. and Villeneuve, A.M. (2004) C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination. Cell 118, 439–452 62 Terada, R. et al. (2002) Efficient gene targeting by homologous recombination in rice. Nat. Biotechnol. 20, 1030–1034 63 Hanin, M. et al. (2001) Gene targeting in Arabidopsis. Plant J. 28, 671–677 64 Iida, S. and Terada, R. (2004) A tale of two integrations, transgene and T-DNA: gene targeting by homologous recombination in rice. Curr. Opin. Biotechnol. 15, 132–138 65 Reiss, B. et al. (2000) RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 97, 3358–3363 66 Jelesko, J.G. et al. (2004) Meiotic recombination between paralogous RBCSB genes on sister chromatids of Arabidopsis thaliana. Genetics 166, 947–957 67 Jelesko, J.G. et al. (1999) Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 96, 10302–10307 68 Christianson, M.L. (1975) Mitotic crossing-over as an important mechanism of floral sectoring in Tradescantia. Mutat. Res. 28, 389–395 69 Burk, L.G. and Menser, H.A. (1964) A dominant aurea mutation in tobacco. Tob. Sci. 8, 101–104 70 Molinier, J. et al. (2004) Interchromatid and interhomolog recombination in Arabidopsis thaliana. Plant Cell 16, 342–352 71 Haubold, B. et al. (2002) Recombination and gene conversion in a 170-kb genomic region of Arabidopsis thaliana. Genetics 161, 1269–1278 72 Culligan, K. et al. (2004) ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell 16, 1091–1104 73 Dubest, S. et al. (2004) Roles of the AtErcc1 protein in recombination. Plant J. 39, 334–342 74 Hanin, M. et al. (2000) Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene. Plant J. 24, 183–189 75 Liu, Z. et al. (2000) Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1. Plant J. 21, 519–528 76 Gallego, F. et al. (2000) AtRAD1, a plant homologue of human and yeast nucleotide excision repair endonucleases, is involved in dark repair of UV damages and recombination. Plant J. 21, 507–518 77 Dubest, S. et al. (2002) Role of the AtRad1p endonuclease in homologous recombination in plants. EMBO Rep. 3, 1049–1054 78 Fidantsef, A.L. et al. (2000) The Arabidopsis UVH1 gene is a homolog of the yeast repair endonuclease RAD1. Plant Physiol. 124, 579–586 79 Costa, R.M. et al. (2001) The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development. Plant J. 28, 385–395 80 Harlow, G.R. et al. (1994) Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation. Plant Cell 6, 227–235 81 Landry, L.G. et al. (1997) An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation. Proc. Natl. Acad. Sci. U. S. A. 94, 328–332 82 Filkowski, J. et al. (2004) Genome stability of vtc1, tt4, and tt5 Arabidopsis thaliana mutants impaired in protection against oxidative stress. Plant J. 38, 60–69 83 Conklin, P.L. et al. (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc. Natl. Acad. Sci. U. S. A. 93, 9970–9974 84 Li, J. et al. (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5, 171–179