Journal of Experimental Botany, Vol. 54, No. 383, pp. 669-680,
February 1, 2003
© 2003 Oxford University Press
Arabidopsis mutants sensitive to gamma radiation include the homologue of the human repair gene ERCC1
Received 16 July 2002; Accepted 2 October 2002
1 Department of Plant Biology, University of California, Davis, Davis, CA 95616, USA
2 Genetics Graduate Group, Department of Plant Biology, University of California, Davis, Davis, CA 95616, USA
3 To whom correspondence should be addressed. E-mail: abbritt{at}ucdavis.edu
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Abstract |
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Mutants sensitive to ionizing radiation in yeast and mammals include an assortment of DNA repair genes. The majority of these DNA repair genes are involved in the repair of DNA double-strand breaks. In this study a forward genetic screen is used to identify
-sensitive mutants of Arabidopsis thaliana. The -plantlet screen used here also reveals two general mutant classes based on size of cotyledons and hypocotyls. One of the mutants discovered is a homologue of the mammalian nucleotide excision repair gene ERCC1. Key words: Arabidopsis, DNA repair, double-strand breaks, ERCC1, ionizing radiation sensitive, Rad10, UV.
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Introduction |
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DNA double-strand breaks (DSBs) present a unique challenge to the cell. In dividing cells unrepaired breaks can initiate cell cycle checkpoints, halting division and, consequently, slowing the growth of the organism (Lydall and Weinert, 1995; Weinert and Lydall, 1998). Furthermore, cells that do divide in the presence of a DSB can produce aneuploid daughter cells that are often not viable (Belyakov et al., 1999; Morgan et al., 1996). Repairing a DSB improperly is potentially more dangerous than not repairing it at all. Improper repair can result in inversions and or translocations that are capable of fostering cancer in mammals (Ferguson and Alt, 2001; Sharpless et al., 2001).
Despite the inherent danger of DSBs, these lesions are sometimes produced and repaired in a programmed fashion. Specialized enzymes create and repair the DSBs required for meiotic recombination and for the generation of immunoglobulins (Grawunder and Harfst, 2001; Keeney et al., 1997; McBlane et al., 1995; Xu and Kleckner, 1995). The replication of nicked templates has recently been recognized as a very significant endogenous source of DSBs. DSBs are also induced, either directly or during the repair process, in the laboratory, by a variety of DNA damaging agents.
The programmed repair of these lesions is facilitated through two dramatically different mechanisms (Jeggo, 1998; Paques and Haber, 1999). In homology dependent repair (HR), the double-stranded gap generated by a frayed DSB is filled by copying, or in some cases splicing, homologous sequences from elsewhere in the genome. Non-homologous end-joining (NHEJ) enables broken ends to be directly reconnected and does not require a homology search, although this process is obviously error prone and degraded or even inappropriate ends may be rejoined. Many, though not all, of the proteins required for HR, NHEJ, or both types of DSB repair have been identified in yeast and mammals (Jackson, 2002; Labhart, 1999; Paques and Haber, 1999; Tsukamoto and Ikeda, 1998).
The study of DSB repair in plants lags far behind that in animals and yeast, but is rapidly accelerating. This interest is largely motivated by the essential roles HR and NHEJ play in the process of genetic engineering. Comparative genomics has uncovered similarities between plants and animals with regard to sequence and complement of repair genes (Arabidopsis Genome Initiative, 2000); the plant complement of recombination-related genes is similar to that of vertebrates (Britt and May, 2003). While knockout mutations of DSB repair genes are generally lethal in mammals, many of these same knockouts are viable in plants, perhaps because plants lack the p53-dependent apoptotic response to the accumulation of DSBs that induces lethality in mammals (Frank et al., 2000). Examples of insertional mutations that are lethal in mammals, but viable in plants, include RAD50 (Gallego et al., 2001; Luo et al., 1999), MRE11 (K Rozwadowski, personal communication; Yamaguchi-Iwai et al., 1999) and, as will be shown in this paper, ERCC1 (Friedberg et al., 1995). The viability of plants defective in these genes makes them an excellent model system for the study of their function not only in DNA repair, but also in meiosis, telomere maintenance, and chromatin remodelling. It also enables a classical genetic hunt to be performed for novel genes required for DSB repair by screening for mutants hypersensitive to DSB-inducing DNA damaging agents. In this paper, a novel screen is described for mutants hypersensitive to gamma () radiation. Of approximately 5000 EMS mutagenized families screened, three were homozygous for recessive mutations that produced both a -hypersensitive but fertile, phenotype. Two of these mutants are both UV- and -sensitive and due to defects in each component of the Arabidopsis homologues of the human endonuclease complex, ERCC1/XPF (a defect which does not result in -sensitivity in mammalian cells). The third mutant is -sensitive, but not UV sensitive; its map position will be described, but its molecular nature has not yet been determined.
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Materials and methods |
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Plant growth conditions
Plants were grown on soil (Sunshine Mix No. 1 or Premier Pro Mix, Premier, Quebec, Canada) sub-irrigated with 0.5x Growmore fertilizer (Growmore, Gardena, CA). Plant growth was carried out in growth chambers maintained at 22 °C under 24 h cool white light filtered through Mylar at a PAR rate of 100 µmol m–2 s–1. Lines to be used for crossing or transformation were grown as stated before except at 16 °C. Plants grown for root length, germination, leaf production, methymethane sulphonate, and mitomycin C assays were grown by placing hypochlorite surface-sterilized seeds on 0.5x Murishige and Skoog media (Sigma-Aldrich, St Louis, MO) supplemented with 0.5x sucrose and solidified with 1% Phytagel. Plates were situated on an aluminium shelf in the growth chamber cooled to 20 °C by Peltier driven cooling blocks (Melcor Trenton, NJ). This reduces lid condensation and plate contamination.
Screen for -sensitive mutants
EMS mutagenized M2 seeds, ecotype Landsberg erecta, were purchased from Lehle Seeds (catalogue M2E-04-05, parental groups 1–5, Tucson AZ). The plants were allowed to self-pollinate and were harvested individually to produce approximately 5000 M3 seed families.
Mutagenized families were screened using the ‘gamma plantlet’ assay for seed sensitivity to ionizing radiation. Seeds from individual families were imbibed and placed in the dark for 24–48 h at 4 °C. Seeds were then removed and irradiated at 10 Krads from a 137Cs irradiator (ITEH, University of California, Davis) at a dose rate of approximately 850 rads min–1. Irradiated and unirradiated siblings of each family were planted side by side on soil in separate pots. Pots were placed in the growth chamber for 12–14 d. After the growth period, pots containing irradiated families were screened for the inability to produce post-embryonic leaves as compared to their unirradiated siblings. Putative -sensitive families were rescreened for sensitivity.
Mapping of -sensitive mutants
M3 individuals from homozygous -sensitive families, ecotype Landsberg erecta (Ler), were outcrossed to wild-type individuals of the Columbia (Col) ecotype. The F1s were self-pollinated to produce segregating F2 seeds. F2 plants were harvested individually to produce F3 seed families. Each family was screened for sensitivity to -radiation. Families homozygous for sensitivity were selected for mapping. Leaf tissue from selected families was harvested for DNA extraction (Quiagen Plant DNA miniprep kit, Valencia, CA). DNA from sensitive lines was used as a substrate for PCR amplification of SSLPs and CAPS (Bell and Ecker, 1994). DNA from Ler, Col or a 50:50 mixture of both DNAs were amplified as controls.
Cloning of mutant and wt alleles
The coding region of AtERCC1 was PCR amplified using the following primers: 5'-CACTCATGGCGAACGAAGAACG-3' and 5'-CCCAAGGAACAAAACCGTG-3'. PCR products were gel purified and cloned into TA cloning vectors (Invitrogen, Carlsbad, CA). Two clones from two separate PCR reactions were amplified and sequenced (DBS sequencing facility, University of California, Davis). Sequences from all four clones were aligned and analysed using Contig Express software (Informax, Bethesda, MD).
Quantification of root sensitivity to UV and
UV-sensitivity assays were performed as reported by Jiang et al. (1997) with the following modifications. The MS plates were supplemented with 0.5% sucrose and solidified with 1% Phytagel (Sigma) instead of Bacto Agar.
Seeds used for -sensitivity assay were surface-sterilized with a 1:3 hypochlorite:water solution containing 0.1% Triton X-100. After sterilization, seeds were suspended in 0.1% agarose and stored at 4 °C in the dark for 3 d to synchronize germination. After day 3 the seeds were removed from the dark and exposed to a dose of 10 Krads by a 137Cs irradiator at a dose rate of approximately 850 rads min–1 (ITEH, University of California, Davis). A second set of samples, that was unirradiated was used as a control. Seeds were sown on plates and the plates were placed in the growth chamber in a vertical position to allow the roots to grow along the surface of the agar. Root length measurements were taken from the bottom of the hypocotyls (identified as the point at which root hairs began) to the tip of the root at 5, 8, and 11 d post-plating for all seedlings that had successfully germinated by the 3rd day post-irradiation. Plants whose roots grew into the agar instead of along the surface were not scored. Scoring was aided by the use of a dissecting microscope.
Quantification of duration of shoot arrest and relative sensitivity to -radiation
For both assays, seeds were surface-sterilized, suspended in 0.1% agarose and stored in the dark at 4 °C for 3 d. Seeds for the shoot assay were either irradiated with 10 Krads as before or were left unirradiated. Seeds were sown on plates and placed in the growth chamber. Seedlings were scored for the presence of post-embryonic leaves 5, 8, 11, 14, and 17 d post-plating with the aid of a dissecting microscope. Seeds that did not germinate within the first 3 d post-plating were not scored.
To determine the relative sensitivity of the mutant versus wild-type lines to -irradiation, seeds were either unirradiated or irradiated with 5, 10, 15, 20, 25, 30, 40 or 50 Krads of -radiation. These seeds were planted on soil and allowed to grow for 14 d. Seedlings were then scored for the presence of post-embryonic leaves. Scoring was performed by eye, scoring organs with trichomes as leaves.
Assay for G2 arrest
A request was made and seeds of Col plants were received that were transformed with a DNA construct (Doerner et al., 1995) and had the promoter for the G2 cyclin B gene driving the transcription of a modified GUS gene that carries a mitotic destruction box. The plants containing this construct are of Arabidopsis ecotype Colombia. These plants were crossed to the -sensitive mutants of this study. Families homozygous for the transformed construct and for -sensitivity were identified by screening individual progenies of F2 plants.
Seeds were sterilized, imbibed and irradiated as before. Irradiated and unirradiated seeds were plated on 0.5x MS Phytogel agar (1%) plates supplemented with 50 µg ml–1 kanamycin. After 6 d seedlings were gathered and stained for the presence of ß-glucuronidase (California Institute of Technology web site: www.its.caltech.edu/
plantlab/protocols/gus.html).
Assay for sensitivity to mitomycin C and methylmethane sulphonate
Mutants were tested for sensitivity to MMC and MMS as previously described (Masson et al., 1997).
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Results |
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The purpose of this study was to identify genes required for the repair of double-strand breaks. Exposure to ionizing radiation produces DSBs (as well as other lesions) in yeast, mammals and flies (Friedberg et al., 1995), and the persistence of these lesions causes cell cycle arrest in yeast and vertebrates (Weinert and Lydall, 1998). Preliminary results suggest that DNA damage-induced cell cycle arrest also occurs in plants. Mutants defective in UVH1, the Arabidopsis homologue of the human XPF (S. cerevisiae RAD1) repair endonuclease component are hypersensitive to the induction of a G2 arrest by ionizing radiation (SB Preuss, AB Britt, unpublished results). This arrest may be the basis of the ‘gamma plantlet’ response, an unusual and long recognized (Haber and Foard, 1961) effect of high doses of ionizing radiation on the post-embryonic development of irradiated seeds. uvh1 is hypersensitive to this effect; its irradiated seeds germinate normally, producing healthy green seedlings at the same rate as unirradiated seeds, but then exhibit a substantial delay in the production of true leaves (Harlow et al., 1994; Jiang et al., 1997).
The sensitivity of the uvh1 repair mutant to the ‘gamma plantlet’ inducing effects of ionizing radiation formed the basis for the screening for additional repair-defective lines. EMS mutagenized M3 families were screened for their inability to produce post-embryonic leaves (true leaves) at 12–14 d post-irradiation of imbibed seeds (Fig. 1). Sibling seeds were irradiated at 10 Krads or unirradiated then sown onto pre-irrigated soil in adjacent pots. A screen of 4167 M3 families produced four confirmed -sensitive families.
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Of the four mutants, three were homozygous for gamma sensitivity, and one appeared to be segregating for sensitivity. Additional M3 sibling seeds of the segregating family were planted and the resulting plants were harvested individually in an attempt to identify a homozygous sensitive individual. Of 25 individually harvested families, 23 displayed a resistant-to-sensitive offspring ratio of 2:1, the remaining two families germinated poorly and were not tested for segregation. The ubiquitous transgenerational expression of an apparently segregating phenotype is probably due to a homozygous mutation that is partially penetrant. Because of the difficulties associated with performing genetics on partially penetrant mutations, this mutant (irs2) will not be discussed further in this paper.
Complementation and backcross
Mutants were backcrossed to their progenitor (Landsberg erecta) line to determine whether they represented single loci, were dominant or recessive, and to reduce the number of secondary mutations in the genetic background. The F1 progeny were selfed and the F2 progeny were scored for -sensitivity as before (Table 1). The expected 3:1 ratio (X2 <3.84) of -resistant-to--sensitive was observed in two of the mutants, consistent with a single locus recessive allele. Initial backcrosses of the third mutant failed and were not reattempted due to findings during complementation testing.
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All three mutants were crossed to one another and to a previously identified
-sensitive mutant uvh1-2 (Harlow et al., 1994), recently determined to be the A. thaliana homologue of the human repair endonuclease factor XPF (Fidantsef et al., 2000; Liu et al., 2000). F2 progeny were scored for sensitivity to -radiation as described before. Most of the crosses produced both -resistant and -sensitive individuals in a ratio of 9:7 (X2 <3.84) (Table 1). However, it was found that one mutant failed to complement uvh1-2, thus representing a new allele of AtXPF1. This mutant was designated uvh1-3. As will be described later, mapping placed one mutant near to a previously identified UV-sensitive mutant uvr7-1 (Jiang et al., 1997). leading to further complementation crosses. Crosses to uvr7-1 failed to complement this mutant. Thus, this mutant was designated uvr7-2. uvr7-1 was not included in the original complementation tests because it displayed only moderate sensitivity to -irradiation in a previous study; the gamma-sensitive phenotype of uvr7-2 is more severe. The third mutant was designated Ionizing Radiation Sensitive 1 (irs1).
Two phenotypically distinct mutant classes
All four mutants exhibit a delay in the production of true leaves after seeds are irradiated with low doses of -radiation (10 Krads). However, the mutants can be divided into two classes based on the length of the hypocotyls and the size of the cotyledons. The first class encompasses mutants irs1 and the partially penetrant mutant irs2. Mutants of this class display shortened hypocotyls and small cotyledons after irradiation of seed. The second class of mutants, encompassing uvr7-2 and uvh1-3, produce normally sized hypocotyls and cotyledons after irradiation (Fig. 2). The class of mutants with small cotyledons and short hypocotyls, irs1 and irs2, is similar in appearance to wild-type irradiated at much higher -doses (40 or more Krads). Conversely, wild-type plants do not display growth characteristics similar to uvr7-2 and uvh1-3 at any -dose.
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Mutant mapping
Two mutants (uvr7-2 and irs1) were mapped by crossing to Arabidopsis ecotype Colombia, selfing the F1 progeny, and characterizing the
-sensitive F2 progeny as to the cosegregation of Landsberg versus Colombia ecotype-specific molecular genetic markers. The mutants are linked to markers on chromosome three (uvr7-2) and chromosome 5 (irs1) (Table 2). The Arabidopsis homologue of the human ERCC1 gene lies in the vicinity of UVR7.
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By scoring recombination events of individual F2 plants for multiple markers, the relative position of the mutant gene with respect to those markers can be determined. Using this approach the position of IRS1 was found to be between markers MDA7 and LFY3. Candidate genes within this interval include plant homologues of human repair genes DNA Ligase IV, XRCC3 and the previously reported Arabidopsis IR-sensitivity locus MIM (Hanin et al., 2000; Mengiste et al., 1999).
Sequence analysis of candidate genes
The candidate gene (AtERCC1) for UVR7 was PCR amplified from genomic DNA, cloned into appropriate vectors and sequenced. Sequences from amplified Landsberg erecta wild type and mutants were compared to identify potential amino acid changes. Sequencing of AtERCC1 from genomic DNA of uvr7-1 and uvr7-2 revealed point mutations that produce a premature stop codon in each allele. The uvr7-1 mutation (highlighted in the sequence gaaaacccaaaaccTagatcgtgattggtg) is a C
T base change resulting in a GLNAMB substitution. This mutation in uvr7-1 truncates the predicted 382 amino acid protein by 358 residues. The uvr7-2 mutation is highlighted within the sequence ttgtgtgcctgAaggtctctgtct is a GA base change resulting in a TRPOPA substitution. This mutation truncates the predicted protein by 158 residues (Fig. 3). Given that uvr7-1 and uvr7-2 fail to complement, and that both mutants contain a premature stop codon within the AtERCC1 gene, it was concluded that the -sensitive phenotypes of uvr7-1 and uvr7-2 are due to mutations in AtERCC1. Surprisingly, the -sensitive phenotype, but not the UV-sensitive phenotype of the longer protein (uvr7-2) is more severe than that of the shorter protein (uvr7-1) (see below).
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Characterization of root and shoot sensitivity to
-radiationThe relative sensitivity of mutants versus Ler to
-radiation was measured in roots and shoots. Using increasing doses of -radiation a dose-response curve was produced for the formation of -plantlets at 14 d post-irradiation (Fig. 4). Mutants uvr7-2 and uvh1 displayed a similar response to -irradiation (6-fold more sensitive than Ler) while irs1 was slightly less sensitive (5-fold more sensitive than Ler) and uvr7-1 was approximately half as sensitive (3-fold more sensitive than Ler). The fold sensitivity was determined by dividing the dose required to yield an average of 1 true leaf in wild type by the dose required to yield an average of 1 true leaf in a mutant line.
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To determine the response of roots to
-irradiation, root growth was measured over time in irradiated versus unirradiated seeds. Root growth was significantly inhibited (versus Ler) by -radiation in all four mutants, again uvr7-1 was significantly less sensitive than uvr7-2 or uvh1. Interestingly, the effects of -radiation on root growth were both more severe and more persistent in irs1 than in the other mutant lines (Fig. 5). A time-course assay for leaf development revealed a similar phenotype with respect to the persistence of the effects of -radiation on the four mutants (Fig. 6). Further observation revealed that many of the irradiated irs1 seedlings became necrotic while irradiated seedlings of other lines displayed little necrosis. The necrosis might be a direct consequence of the failure to produce new leaves, or an indirect consequence of the nearly complete failure of root development in the irradiated seedlings.
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Meristematic cell cycle arrest in response to damage
One explanation for the failure to produce non-embryonic leaves and the reduced root growth is that meristematic cells of the root and shoot undergo cell cycle arrest in response to damage. To test this hypothesis, a DNA construct containing a G2-specific promoter driving a modified ß-glucuronidase gene carrying a mitotic destruction box was crossed into mutant lines uvr7-2, uvh1, and irs1. The G2-specific expression of ß-glucuronidase (subsequently degraded at mitosis) allowed for the visualization of cells progressing through or arrested in G2 (Colon-Carmona et al., 1999).
Seedlings were collected at 6 d post-irradiation and stained for the presence of ß-glucuronidase. Seedlings from irradiated uvr7-2 and uvh1 accumulated ß-glucuronidase in meristem cells, visualized as blue staining, suggesting that the cells are arresting in G2. In contrast, seedlings from irradiated irs1 seeds presented only a few blue cells, similar to their unirradiated siblings (Fig. 7).
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Mutant sensitivity to additional damaging agents
To determine possible damage repair pathway overlap of these mutants, sensitivity to a variety of DNA damaging agents was tested. Ultraviolet radiation (UV), methylmethane sulphonate (MMS) and mitomycin C (MMC) are known DNA damaging agents that have little overlap in their damage spectrum. UV creates pyrimidine dimers, MMS is an alkylating agent that adds methyl groups to a variety of sites and MMC is a interstrand DNA crosslinking agent (Friedberg et al., 1995).
Mutant irs1 displayed little or no sensitivity to UV, MMS or MMC, suggesting that irs1 is defective in a highly specific repair pathway for damage created by ionizing radiation, perhaps double-strand breaks (Figs 8, 9).
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In humans and yeast, ERCC1 (yeast Rad10) complexes with XPF (yeast Rad1) to form a structure-specific endonuclease. This enzyme is involved in the removal of pyrimidine dimers through the nucleotide excision repair pathway, and DNA interstrand crosslinks through an undefined pathway (de Laat et al., 1998; De Silva et al., 2000). Not unexpectedly, mutant uvr7-2 is highly sensitive to MMC and UV, and is also slightly sensitive to MMS (Figs 8, 9).
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Discussion |
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The ultimate goal of this study was to identify genes that are involved in recombination in plants. The isolation of mutants hypersensitive to the lethal effects of IR has proved useful for the identification of genes required for DSB repair in yeast and mammals (Paques and Haber, 1999; van Gent et al., 2001). A screen for IR-sensitive mutants in plants might, therefore, reveal the genes required for DSB repair. It should be noted, however, that IR, in addition to generating DSBs, induces a wide variety of types of damage to both DNA and other cellular constituents. For example, a recent genome-wide screen of yeast deletion mutations has revealed an additional 107 required for gamma radiation resistance (Bennett et al., 2001). Although many of these genes appear to be involved in repair, chromatin maintenance or cell cycle response, there are also genes predicted or known to be involved in a wide variety of other cell functions. Previous screens for IR-sensitive Arabidopsis mutants (identified as exhibiting enhanced sensitivity to the inhibitory effects of
-radiation on root elongation (Masson and Paszkowski, 1997) have produced valuable and interesting mutations affecting stress response, gene silencing, and meiotic recombination rate. However, the connection of these mutations to DNA repair remains obscure, and molecular analysis of the mutations has not revealed lesions in the Arabidopsis homologues of genes known to be required for DSB repair in other organisms. For this reason, an attempt was made to develop a screen for -sensitive mutants that does not cast quite so wide a net.
In plants, ionizing radiation has immediate and distinguishable effects both on cell expansion and cell division. Harlow et al. (1994) made the original observation that -irradiation of uvh1 Arabidopsis mutant seeds could inhibit organogenesis (which requires cell division, as well as cell expansion) without inhibiting the rate of germination or the expansion of pre-existing embryonic tissues (processes that are largely driven by cell expansion). In other words, irradiated seeds germinated normally, but exhibited a substantial delay (two weeks or more, depending on dose) in the production of true leaves. Similarly, Jiang et al. (1997) observed that the roots of uvh1 seedlings are no more sensitive to the short-term effects of ionizing radiation on root elongation (primarily a function of cell expansion) than their Ler progenitors. It was hypothesized that this differential sensitivity to the effects of -radiation on cell division versus cell expansion might be due to the induction of a cell cycle checkpoint by the persistence of a DNA damage product that would normally be repaired via a UVH1-dependent pathway and, indeed, the meristematic cells of this line do accumulate in G2 after irradiation (SB Preuss, unpublished results).
UVH1 encodes the Arabidopsis homologue of the human XPF protein (Fidantsef et al., 2000; Liu et al., 2000). The XPF (in S. cerevisiae, RAD1) protein forms half of the repair endonuclease heterodimer required for nucleotide excision repair (NER) of damaged bases and is involved in the repair of other splayed DNA structures such as the flaps formed during non-homologous end-joining of DSBs (Fig. 0
). Because other Arabidopsis nucleotide excision repair mutants are not especially sensitive to any of the effects of -radiation (Jiang et al., 1997) it was reasoned that the sensitivity of the uvh1 mutant to the -plantlet-inducing effects of radiation must be due to a role in the repair of a critical -induced lesion, such as a cross-linked or broken chromosome, rather than due to its role in NER.
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Here the ‘gamma plantlet’ effect was tested as a basis for screening for mutants defective in the repair of checkpoint-inducing lesions. Seeds of EMS-mutagenized M3 families (progenies of individual M2 plants, which may be homozygous for new mutations) were irradiated with doses of
-radiation that do not affect the germination or development of the progenitor line. Families were selected that produced -plantlets (otherwise healthy plants that fail to produce true leaves by day 14 when irradiated with normally innocuous doses of ). Control, unirradiated seedlings were also grown for comparison. Of over 4000 families screened, only four produced this -dependent phenotype. One of those families was segregating for the phenotype and was later determined to be homozygous for a partially penetrant mutation (this mutation was termed irs2). The remaining three mutants were fertile and phenotypically normal in the absence of radiation, represented single locus recessive mutations, and fell into three complementation groups. One of the mutants represented a third allele of UVH1, the Arabidopsis homologue of the human XPF repair endonuclease factor described above. Given the fact that this screen was developed based on the uvh1 phenotype, the reisolation of a mutation in this gene is not surprising, although it does suggest that there may be a limited number of genes that can be mutated to produce this phenotype. The nature of the base change that forms this allele has not yet been determined; its identification as an allele of UVH1 is based on complementation tests.
A second mutation proved to be a nonsense mutation in the Arabidopsis homologue of the human ERCC1 gene. The isolation of a mutation in this gene is consistent with the notion that, in plants, as in other living things, ERCC1 and XPF form the heterodimer that constitutes a repair endonuclease. It was also found that this mutation was allelic to the previously identified mutant uvr7-1, a mutant that, although more sensitive to -radiation than other NER-defective mutants, was not as sensitive to -radiation as uvh1-2 (Jiang et al., 1997). A side-by-side comparison of the alleles uvr7-1 and uvr7-2 indicates that while both mutants are more sensitive to -radiation than wild type, they do differ significantly in their sensitivity to radiation (Figs 4, 5, 6). By contrast, they are identical in their sensitivity to UV (Fig. 8). It is interesting that the allele with the less severe phenotype (uvr7-1) contains the more severe mutation; the predicted protein is only 22 amino acids long (Fig. 3). By contrast, the uvr7-2 allele is predicted to make a protein 224 amino acids in length (about 2/3 of the wild-type protein); perhaps this putative protein fragment is interacting with other repair factors in a manner that inhibits their function, or to a DNA lesion specific to -irradiation. However, one must take note that the mutation is recessive, not dominant. In the presence of the wild-type allele the uvr7-2 allele has no obvious deleterious effect on IR resistance.
In the absence of exogenous DNA damaging agents, AtERCC1 knockouts are phenotypically normal. In contrast, ERCC1 mutations in mammals are lethal. Homozygous mouse knockouts die before weaning, apparently of liver failure, although many organs are compromised (Selfridge et al., 2001; Weeda et al., 1997). The cells of affected tissues in mouse ERCC1 knockouts become highly aneuploid and polyploid, perhaps in response to some sort of endogenous damage that cannot be repaired in the absence of ERCC1.
Although mammalian XPF or ERCC1-defective cell lines share the Arabidopsis mutant’s sensitivity to the cross-linking agent MMC, mammals apparently lack the -sensitive phenotype of plants defective in XPF and ERCC1. The XPF/ERCC1 endonuclease clearly plays a role in some mammalian recombination processes and in the recombinational repair of certain lesions; for example, the production of UV-induced rearrangements requires both of these proteins (Chipchase and Melton, 2002), and the endonuclease is also required for the integration of transgenes (Niedernhofer et al., 2001). But cell lines defective in these genes are no more sensitive to the lethal effects of ionizing radiation than wild-type lines. This difference between plants and animals might reflect a profound difference in the repair pathways for IR-induced damage; it is possible that plants are relying heavily on the XPF/ERCC1 endonuclease to process some type of damage (or some repair intermediate) that is efficiently handled by another pathway in animals. Alternatively, it is possible that plant and animal responses to various lesions differ. Animals actively induce a p53-dependent apoptotic response to the accumulation of certain types of damage; plants lack a p53 homologue and may lack a functionally homologous response. This assay for -sensitivity is not an assay for cell death, it is an assay for the arrest of cell division. It is possible that mammalian cells assayed for -sensitivity are dying due to a response to the accumulation of some -induced lesion that activates an apoptotic pathway, and that plants lack this apoptotic response. This viability would then make it possible for a cell cycle response to be observed (here G2 arrest, Fig. 7) that simply might not exist, or cannot be observed, in suicidal mammalian cells.
The screen for induction of -plantlets at low IR doses actually produced two distinct classes of phenotypes. At a dose to seeds of 10 Krad, atercc1 and atxpf mutants produce very healthy seedlings, with well-expanded cotyledons, that transiently experience a G2 arrest and then go on to produce true leaves, although there are some changes in leaf morphology and phyllotaxy (not shown). It is stressed here that this response does not mimic the response of wild-type plants to higher doses of -radiation. Gamma doses high enough to produce -plantlets from wild-type Arabidopsis seeds also result in the formation of tiny cotyledons and shorter hypocotyls. These stunted wild-type plants express anthocyanins, necrose, and, if they survive, produce very tiny malformed plants.
The irs1 mutant mimics the behaviour of wild-type gamma plantlets, though at reduced doses (10 Krads versus 40 Krads to produce 100% gamma plantlets). Cotyledons do not expand (Fig. 2), and the roots are stunted and do not recover (Fig. 5). There is no obvious G2 arrest. Also in contrast to atercc1 and atxpf, there is no sensitivity to UV or MMC.
The molecular/biochemical basis of the irs1 mutant remains to be determined. Mapping of irs1 places the mutant gene in a region of chromosome 5 that encompasses Arabidopsis homologues of the human DNA repair genes DNA Ligase IV and XRCC3. This region also contains an SMC (structural maintenance of chromosomes) gene MIM. Mutants of these genes in their respective organisms are sensitive to ionizing radiation.
Of the three genes listed above it may be possible to rule out MIM as a candidate for irs1, based on mutant phenotypes other than sensitivity to ionizing radiation. While mim is sensitive to MMS and UV-C, irs1 is not. In addition, roots of untreated mim seedlings are deficient in elongation at 10 d post-plating while irs1 appears wild-type when observed in a similar manner. Although evidence suggests that irs1 and mim are not alleles of the same gene, complementation will be carried out to account for this possibility.
Recombination-related DNA repair pathways are involved in not only repair, but in mutagenesis, in the production of genetic diversity, in stress tolerance, and in both traditional and transgenic plant breeding technologies. As such, they are obviously of interest to applied and basic plant geneticists, and the identification of genes required for these processes is a critical step in the development of techniques required for the more precise and predictable genetic engineering of crops. Although reverse genetic approaches have just begun to enhance an understanding of these processes, plants may differ from animals in some of their strategies for repair, and not all genes involved in recombination have been identified in other model systems. The continued identification of plant DNA repair mutations, such as the one in AtERCC1 described above, suggest that classical genetic analysis of IR-sensitive mutants in Arabidopsis will continue to contribute to understanding genetic recombination in plants. Indeed, plants’ enhanced tolerance for persisting DNA damage may make them an ideal system for the identification of genes not accessible to classical genetic analysis in mammalian model systems.
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Acknowledgements |
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The authors greatly appreciate Samantha Barling-Silva’s assistance in producing the figures for this paper. We also thank our fellow laboratory members, Kevin Culligan, Joanna Friesner, and James Hatteroth for providing useful suggestions during the course of this work. This research was sponsored by the United State–Israel Binational Agricultural Research Fund (project No. US-3223-01C) and the National Science Foundation (Grant No. MCB-9983142).
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Allard RW. 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24, 235–278.
Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.[CrossRef][Medline]
Araujo SJ, Wood RD. 1999. Protein complexes in nucleotide excision repair. Mutation Research 435, 23–33.[Web of Science][Medline]
Bell CJ, Ecker JR. 1994. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19, 137–144.[CrossRef][Web of Science][Medline]
Belyakov OV, Prise KM, Trott KR, Michael BD. 1999. Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or alpha-particles. International Journal of Radiation Biology 75, 985–993.[CrossRef][Web of Science][Medline]
Bennett CB, Lewis LK, Karthikeyan G, Lobachev KS, Jin YH, Sterling JF, Snipe JR, Resnick MA. 2001. Genes required for ionizing radiation resistance in yeast. Nature Genetics 29, 426–434.[CrossRef][Web of Science][Medline]
Britt AB, May G. 2003. Re-engineering plant gene targeting. Trends in Plant Science (in press).
Chipchase MD, Melton DW. 2002. The formation of UV-induced chromosome aberrations involves ERCC1 and XPF but not other nucleotide excision repair genes. DNA Repair 1, 335–340.[Medline]
Colon-Carmona A, You R, Haimovitch-Gal T, Doerner P. 1999. Spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. The Plant Journal 20, 503–508.[CrossRef][Web of Science][Medline]
de Laat WL, Appeldoorn E, Jaspers NGJ, Hoeijmakers JHJ. 1998. DNA structural elements required for ERCC1-XPF endonuclease activity. Journal of Biological Chemistry 273, 7835–7842.
De Silva IU, McHugh PJ, Clingen PH, Hartley JA. 2000. Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Molecular and Cellular Biology 20, 7980–7990.
Doerner P, Carmona AC, Jorgensen J-E, Steppuhn J, Lamb C. 1995. Cell cycle regulation during root organogenesis. Journal of Cellular Biochemistry Supplement 0, 129.
Ferguson DO, Alt FW. 2001. DNA double-strand break repair and chromosomal translocation: lessons from animal models. Oncogene 20, 5572–5579.[CrossRef][Web of Science][Medline]
Fidantsef A, Mitchell D, Britt A. 2000. The Arabidopsis UVH1 gene is a homologue of the yeast repair endonuclease RAD1. Plant Physiology 124, 579–586.
Frank KM, Sharpless NE, Gao Y, Sekiguchi JM, Ferguson DO, Zhu C, Manis JP, Horner J, DePinho RA, Alt FW. 2000. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Molecular Cell 5, 993–1002.[CrossRef][Web of Science][Medline]
Friedberg EC, Walker GC, Siede,W. 1995. DNA repair and mutagenesis. Washington, D.C: ASM Press.
Gallego ME, Jeanneau M, Granier F, Bouchez D, Bechtold N, White CI. 2001. Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity. The Plant Journal 25, 31–41.[CrossRef][Web of Science][Medline]
Grawunder U, Harfst E. 2001. How to make ends meet in V(D)J recombination. Current Opinion in Immunology 13, 186–194.[CrossRef][Web of Science][Medline]
Haber AH, Foard DE. 1961. Anatomical analysis of wheat growing without cell division. American Journal of Botany 48, 438–446.[CrossRef][Web of Science]
Hanin M, Mengiste T, Bogucki A, Paszkowski J. 2000. Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene. The Plant Journal 24, 183–189.[CrossRef][Web of Science][Medline]
Harlow GR, Jenkins ME, Pittalwala TS, Mount DW. 1994. Isolation of Uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation. The Plant Cell 6, 227–235.[Abstract]
Jackson SP. 2002. Sensing and repairing DNA double-strand breaks. Carcinogenesis (Oxford) 23, 687–696.
Jeggo PA. 1998. Identification of genes involved in repair of DNA double-strand breaks in mammalian cells. Radiation Research 15, S80–S91.[CrossRef]
Jiang C-Z, Yen C-N, Cronin K, Mitchell D, Britt A. 1997. UV- and gamma-radiation sensitive mutants of Arabidopsis. Genetics 147, 1401–1409.[Abstract]
Keeney S, Giroux CN, Kleckner N. 1997. Meiosis-specific DNA double-strand breaks are catalysed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384.[CrossRef][Web of Science][Medline]
Labhart P. 1999. Non-homologous DNA end joining in cell-free systems. European Journal of Biochemistry 265, 849–861.[Web of Science][Medline]
Liu Z, Hossain GS, Islas-Osuna MA, Mitchell DL, Mount DW. 2000. Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homologue of Saccharomyces cerevisiae Rad1. The Plant Journal. 21, 519–528.[CrossRef][Web of Science][Medline]
Luo G, Yao MS, Bender CF, Mills M, Bladl AR, Bradley A, Petrini JHJ. 1999. Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proceedings of the National Academy of Sciences, USA 96, 7376–7381.
Lydall D, Weinert T. 1995. Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270, 1488–1491.
Masson J, Paszkowski J. 1997. Arabidopsis thaliana mutants altered in homologous recombination. Proceedings of the National Academy of Sciences, USA 94, 11731–11735.
Masson JE, King PJ, Paszkowski J. 1997. Mutants of Arabidopsis thaliana hypersensitive to DNA-damaging treatments. Genetics 146, 401–407.[Abstract]
McBlane JF, Van Gent,DC, Ramsden DA, Romeo C, Cuomo CA, Gellert M, Oettinger MA. 1995. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395.[CrossRef][Web of Science][Medline]
Mengiste T, Revenkova E, Bechtold N, Paszkowski J. 1999. An SMC-like protein is required for efficient homologous recombination in Arabidopsis. EMBO Journal 18, 4505–4512.[CrossRef][Web of Science][Medline]
Morgan WF, Day JP, Kaplan MI, McGhee EM, Limoli CL. 1996. Genomic instability induced by ionizing radiation. Radiation Research 146, 247–258.[Web of Science][Medline]
Niedernhofer LJ, Essers J, Weeda G, Beverloo B, de Wit J, Muijtjens M, Odijk H, Hoeijmakers JHJ, Kanaar R. 2001. The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells. EMBO Journal 20, 6540–6549.[CrossRef][Web of Science][Medline]
Paques F, Haber JE. 1999. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 63, 349–404.
Selfridge J, Hsia K-T, Redhead NJ, Melton DW. 2001. Correction of liver dysfunction in DNA repair-deficient mice with an ERCC1 transgene. Nucleic Acids Research 29, 4541–4550.
Sharpless NE, Ferguson DO, O’Hagan RC, et al. 2001. Impaired non-homologous-end-joining provokes soft tissue sarcomas harboring chromosomal translocations, amplifications, and deletions. Molecular Cell 8, 1187–1196.[CrossRef][Web of Science][Medline]
Tsukamoto Y, Ikeda H. 1998. Double-strand break repair mediated DNA end-joining. Genes to Cells 3, 135–144.[Abstract]
van Gent DC, Hoeijmakers JHJ, Kanaar R. 2001. Chromosomal stability and the DNA double-stranded break connection. Nature Reviews Genetics 2, 196–206.[CrossRef][Web of Science][Medline]
Weeda G, Donker I, De Wit J, Morreau H, Janssens R, Vissers CJ, Nigg A, Van Steeg H, Bootsma D, Hoeijmakers JHJ. 1997. Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence. Current Biology 7, 427–439.[CrossRef][Web of Science][Medline]
Weinert T, Lydall D. 1998. Pathways and puzzles in DNA replication and damage checkpoints in yeast. 363–382.
Xu L, Kleckner N. 1995. Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast. EMBO Journal 14, 5115–5128.[Web of Science][Medline]
Yamaguchi-Iwai Y, Sonoda E, Sasaki MS, et al. 1999. Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells. EMBO Journal 18, 6619–6629.[CrossRef][Web of Science][Medline]
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