The Plant Journal (2009) 60, 509–517 doi: 10.1111/j.1365-313X.2009.03974.x A UVB-hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage response Ayako N Sakamoto1,*, Vo Thi Thuong Lan1,†, Vichai Puripunyavanich1,‡, Yoshihiro Hase1, Yuichiro Yokota1, Naoya Shikazono1,2, Mayu Nakagawa1, Issay Narumi1 and Atsushi Tanaka1 Radiation-Applied Biology Division, Japan Atomic Energy Agency, Watanuki-machi 1233, Takasaki, Gumma 370-1292, Japan, and Advanced Science Research Center, Japan Atomic Energy Agency, 2–4 Shirakata-Shirane, Tokai, Ibaraki 319-1195, Japan Received April 2009; revised 20 June 2009; accepted 29 June 2009; published online 11 August 2009 * For correspondence (fax +81 27 346 9688; e-mail sakamoto.ayako@jaea.go.jp) † Present address: Hanoi University of Science, Hanoi, Vietnam ‡ Present address: Thailand Institute of Nuclear Technology, Bangkok, Thailand SUMMARY To investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to c-radiation and hydroxyurea This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1 RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast twohybrid system, suggesting that the proteins form a homodimer Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after c-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP Keywords: checkpoint, cell cycle, ultraviolet light, ATR, DNA damage, hydroxyurea INTRODUCTION Plants are continuously exposed to environmental stresses that may damage the integrity of the genome For example, the UVB (290–320 nm) component of sunlight induces various types of DNA damage, such as formation of cyclobutane pyrimidine dimers (CPD) or 6-4 photoproducts (6-4PP), which prevent DNA replication and/or cell proliferation, and cause mutations (Britt, 1999) Plants encodes various DNA repair enzymes, e.g photolyases (Ahmad et al., 1997; Nakajima et al., 1998), DNA endonucleases (Gallego et al., 2000; Liu et al., 2001) or DNA glycosylases (Gao and Murphy, 2001; Garcı´a-Ortiz et al., 2001), that remove the DNA damage and support continuous growth in harmful environments To survive in a stressful environment, it is also important for plants to coordinate DNA-repair activities with cell-cycle ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd progression Unrepaired DNA damage occurring in S phase can result in mutagenic replication, while incomplete DNA repair prior to M phase can cause chromosome loss Many eukaryotes have ‘cell-cycle checkpoint’ systems that verify whether the processes at each phase of the cell cycle have been accurately completed before progression to the next phase The majority of these checkpoint responses, which appear to be highly conserved throughout eukaryotes, are triggered by activation of two major sensor proteins, ATM (ataxia-telangiectasia-mutated) and ATR (ataxia-telangiectasia- and Rad3-related) ATM primary responds to DNA double-strand breaks, whereas ATR primary responds to DNA damage or agents that block DNA replication The checkpoint signals are then transferred to mediator proteins, 509 510 Ayako N Sakamoto et al such as checkpoint kinase or (CHK1 or CHK2), through protein phosphorylation cascades that directly regulate cell-cycle progression Homologues of ATM and ATR have been previously identified in Arabidopsis thaliana (Garcia et al., 2000; Culligan et al., 2004) Mutant atm plants show hypersensitivity to c-rays, but not to UVB, aphidicolin or hydroxyurea (HU), suggesting functional conservation of the ATM-dependent pathway in higher plants (Garcia et al., 2003) Similarly, atr mutants show hypersensitivity to all of these agents, which is reminiscent of the phenotype of ATR-deficient mammalian cells (Culligan et al., 2004) These results suggest that the function of ATR is conserved in higher plants, at least in part Interestingly, however, the atr mutant plants are viable, in contrast to ATR knockout mice Accumulation of CYCB1;1:GUS fusion protein in the c-ray-irradiated wild-type plants but not in the ATR knockout plant suggested that the checkpoint signal regulated by ATR is transferred to the cyclin/CDK complex (through the ATR activity) in Arabidopsis (Culligan et al., 2006) In Arabidopsis, a downstream target of AtATM kinase, AtNBS1, has been identified, and is suggested to be involved in the signal transduction pathway triggered by double-strand breaks (Waterworth et al., 2007) However, there is little information about the pathway downstream of AtATR, as homologues of neither CHK1 nor CHK2 have been found in Arabidopsis so far Thus, the ATR-mediated checkpoint signal pathway is largely unknown in higher plants Here we report the isolation and characterization of a novel UVB-, c-ray- and HU-sensitive mutant suv2 (sensitive to UV 2) The gene disrupted in suv2 is predicted to encode a protein that displays similarity to mammalian ATR-interacting protein (ATRIP) or fission yeast Rad26 The presence of the potential target sequence of phosphoinositole-3-kinaserelated protein kinases (PIKKs) revealed that this protein could be a substrate of ATR Thus, we named this gene AtATRIP Further detailed characterization of the AtATRIP protein could provide novel information of the checkpoint pathway in higher plants RESULTS AND DISCUSSION The suv2-1 mutant is hypersensitive to DNA-damaging agents To isolate novel genes involved in the UV response in higher plants, approximately 3000 M2 lines of ion beamirradiated Arabidopsis plants were screened for UVB hypersensitivity under dark conditions (Sakamoto et al., 2003) Six independent mutants isolated from this screening were tentatively named suv (sensitive to UV) The suv2-1 mutation, the focus of this paper, mapped to the bottom of chromosome 5, and was a single-site recessive mutation (data not shown) To determine the function of the gene disrupted in suv2-1, we exposed the mutant plants to various DNA-damaging agents and observed their responses Three-day-old seedlings were exposed to various doses of UVB or c-rays, or transplanted to new plates supplemented with various concentrations of mitomycin C (MMC) Root growth after exposure/transplantation was then measured and plotted When the plants were exposed to UVB, the root growth of suv2-1 plants was more severely inhibited than that of wildtype plants under both dark and light conditions (Figure 1a,b) Similarly, the c-ray exposure and MMC treatment had more severe effects on suv2-1 plants than on wild-type plants (Figure 1c,d) These results suggest that the gene disrupted in suv2-1 is involved in a pathway responding to various kinds of DNA damage rather than specific DNArepair pathways such as photorepair or excision-repair pathways Identification of the gene responsible for the suv2 mutation To identify the mutation present in suv2-1, we performed fine mapping by the SSLP or CAPS methods Approximately 300 UVB-sensitive F2 lines derived from a cross between suv2-1 and Ler were investigated Among the markers examined, markers on K2N11, MRA19 and K15I22 showed no recombination in 608 chromosomes, while markers on K9E15 or MCL19 showed one or two recombination events (Figure 2a) As ion-beam irradiation often induces large chromosome rearrangements such as inversions and/or translocations (Sakamoto et al., 2003; Kitamura et al., 2004; Shikazono et al., 2005; Rahman et al., 2006), we first examined whether the chromosome region surrounding these markers showed a large structural change We obtained the P1 clones MRA19 and MCL19 from Kazusa DNA Research Institute and BAC clone K15I22 from the Arabidopsis Biological Resource Center (Figure 2a), and used the whole P1 or BAC DNA as a probe for genomic DNA blotting Probe MRA19 detected a band of approximately 5.3 kb that was present in the wild-type but not in suv2-1 (Figure 2b,e) Four pairs of PCR primers covering the 5.3 kb fragment were designed to test whether each sub-fragment was amplified or not One primer pair failed to amplify the expected band, suggesting that the corresponding region was altered in the suv2-1 (Figure 2c) To identify the exact position of the rearrangement, TAIL-PCR was performed using nine sets of specific (SP) primers (Figure S1) and arbitrary degenerated (AD) primers Sequencing analysis of TAIL-PCR products revealed the structure of the chromosomal rearrangement that occurred in suv2-1 In particular, the suv2-1 chromosome had an insertion within the region corresponding to MRA19 (Figures 2d and 3, and S1) The inserted DNA appears to have originated from two sites on chromosome 5: a 1114 bp fragment from MHJ24 and a 996 bp fragment from MDC12 were fused and inserted into the region corresponding to MRA19 PCR analysis revealed that ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 Novel checkpoint gene in Arabidopsis 511 (a) (b) yeast Rad26, both of which are involved in the damage checkpoint pathway (Figure 3b) Phylogenic analysis of this gene product and other ATRIP/Rad26 family proteins suggests a close relationship between them (Sweeney et al., 2009) MRA19.1 cDNA complements the suv2 mutants (c) (d) Figure Sensitivity of suv2-1 to UVB, c-rays and MMC Three-day-old seedlings were irradiated with UVB (a, b) or c-rays (c) or transplanted onto new plates supplemented with MMC (d) The seedlings were grown for another days under continuous white light (a, c, d) or in the dark (b) The root growth after irradiation or transplantation was measured using NIH Image, and expressed as a percentage of the mean length of nonirradiated wild-type roots or wild-type roots on non-supplemented plates Each value is the mean Ỉ SD of 15–25 measurements the authentic chromosome regions corresponding to MHJ24 and MDC12 were intact in suv2-1 (Figure 2d) This is probably due to duplication of DNA fragments during the repair process after ion-beam irradiation The chromosome rearrangement in suv2-1 disrupted a gene annotated as MRA19.1 (At5g45610) The MRA19.1 gene originally annotated consisted of 11 exons, encoding an unknown protein To confirm the nucleotide sequence of MRA19.1 transcripts, we performed RT-PCR with total RNA from wild-type plants, and determined the cDNA sequence As a result, we found that the MRA19.1 transcript had an additional short exon between the originally predicted exon and exon The resulting new ORF consists of 12 exons encoding a 646 amino acid protein (Figure 3a) A homology search with the amino acid sequence indicated no significant homology to any previously reported proteins However, analysis using a coiled-coil region prediction program (Lupas et al., 1991) revealed that the region surrounding the novel exon had a high score for coiledcoil structure Helical wheel analysis of the amino acid sequence predicted a typical amphipathic helix structure in this region, which could make a solid protein–protein interaction surface (data not shown) In addition, the amino acid sequence contained two SQ sequences that are known targets of PIKK kinases These characteristics are reminiscent of mammalian ATR-interacting protein ATRIP or fission To confirm whether the disruption of the MRA19.1 gene is responsible for the suv2 phenotype, we studied another mutant line suv2-2 (SALK_077978; Figure 3a) We examined the UVB sensitivity of suv2-1, suv2-2 and their F1 progeny (suv2-1/suv2-2) We found that the root growth of the suv2-1, suv2-2 and suv2-1/suv2-2 plants was indistinguishable from each other, and that these roots were shorter than those of the wild-type plants (data not shown) To confirm whether the isolated MRA19.1 cDNA includes a full ORF and is sufficient to complement the suv2 phenotype, the cDNA was introduced into the suv2-2 lines under the control of the 35S promoter The homozygous transgenic 35S:MRA19.1 line was isolated and examined for UVB hypersensitivity by a root-bending assay (see Experimental procedures) Root growth of the transgenic plants was indistinguishable from that of wild-type plants, and was significantly greater than that of the parental suv2-2 plants This result suggests that the MRA19.1 cDNA is functional and sufficient to complement the UVB sensitivity of the suv2 mutant (Figure 4) According to publicly available microarray data [for example, the Arabidopsis eFP browser (http://bar.utoronto ca/efp/cgi-bin/efpWeb.cgi) or Genevestigator (https://www genevestigator.com/gv/user/serveApplet.jsp)], the MRA19.1 gene (At5g45610) is expressed constantly throughout the developmental stages and in various tissues When 3-day-old seedlings were exposed to 100 Gy of c-rays or kJ m)2 of UVB, the MRA19.1 mRNA level was slightly increased (data not shown and Figure S2c) In addition, long-term UVB irradiation of 3-week-old plants also increased the MRA19.1 mRNA level (Figure S2c) suv2 is reminiscent of the atr mutant The characteristics of the MRA19.1 protein suggests that this gene may be involved in cell-cycle checkpoint regulation and possibly associated with AtATR in Arabidopsis To compare the phenotype of AtATR-disrupted plants with that of suv2, we obtained a T-DNA insertion mutant of ATR (atr-5; SALK_083543) Semi-quantitative RT-PCR analysis revealed that the level of AtATR transcripts was strongly reduced in atr-5 (Figure 2), suggesting that the AtATR gene is inactivated in this line First, we examined the responses of atr-5 and suv2-1 plants to HU When the suv2-1 seedlings were transplanted onto plates including 0.25 or 0.5 mM HU, the root growth of suv2-1 seedlings was severely inhibited compared to wildtype plants (Figure 5) The growth inhibition level of suv2-1 ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 512 Ayako N Sakamoto et al (a) (b) (c) (d) (e) was very similar to that of atr-5 plants Moreover, the growth of an atr-5 suv2-1 double mutant was indistinguishable from that of suv2-1 and atr-5 single mutants In addition, the suv21 plants were hypersensitive to aphidicolin, similar to atr-5 plants (data not shown) These results are consistent with the possibility that the gene disrupted in suv2 is involved in the same pathway as AtATR for tolerance of replication inhibitors To investigate the possible roles of the gene disrupted in suv2, we introgressed the CYCB1;1:GUS gene construct, a marker of G2/M progression (Colo´n-Carmona et al., 1999), into the suv2-1 and atr-5 backgrounds Transgenic plants in wild-type, suv2-1 or atr-5 background were treated with crays, and GUS expression was observed 2–24 h after treatment When the wild-type background plants were treated with 100 Gy of c-rays, several cells in the root tip started to accumulate CYCB1;1:GUS protein at h, resulting in spotted patterns of GUS staining (Figure 6a) At h, the GUS staining became uniform and spread to throughout the root tip This staining pattern was still seen even at 24 h, suggesting G2 cell-cycle arrest in almost all cells in the Figure Mapping analysis and detection of the chromosomal rearrangement (a) Mapping analysis of the suv2-1 mutation suv2-1 was mapped to the bottom of chromosome An approximately 700 kb region of chromosome (positions 18 300 000– 19 000 000) and major BACs (filled arrows) are shown, as well as the recombination frequencies (numbers) No recombination was observed for the K2N11, MRA19 and K15I22 markers (b) Restriction map of MRA19 The vertical lines show the restriction sites for EcoO109 I The numbers show the size of major fragments The 5.3 kb fragment that was absent in the Southern blotting analysis with suv2-1 DNA was analyzed further (c) PCR analysis of suv2-1 The horizontal and vertical lines show the 5.3 kb fragment and the restriction sites of EcoO109 I, respectively The horizontal arrows indicate the two annotated ORFs present in this region The filled rectangles indicate the regions amplified by PCR with suv21 DNA The open rectangle indicates the region that was not amplified by PCR with suv2-1 DNA (d) Chromosome rearrangement in the suv2-1 mutant Two DNA fragments from the regions corresponding to MDC12 and MHJ24 were duplicated and inserted into a site in MRA19 Arrows indicate the direction of the fragments The diagram is not drawn to scale (e) Southern blot analysis with wild-type (Col) and suv2-1 DNA The genomic DNA was digested with EcoO109 I and hybridized with a probe prepared from P1 clone MRA19 The 5.3 kb band (asterisk) present in the Col DNA was absent in suv2-1, and a band of approximately kb appeared instead (arrow) wild-type root tip at 24 h post-irradiation In contrast, the staining patterns in the suv2-1 and atr-5 background were significantly weaker than those in the wild-type background (Figure 6a) The staining in suv2-1 and atr-5 roots was reduced 6–24 h after the c-ray treatment when wild-type roots still showed uniform staining These results suggest that many of the cells in the suv2-1 and atr-5 root meristems failed to arrest in G2 in the presence of double-strand breaks In addition, we observed the GUS expression patterns of aphidicolin-treated plants Similar to c-radiation treatment, the suv2-1 and atr-5 plants displayed less overall GUS staining than observed in wild-type plants (Figure 6b) Taken together, these results strongly support the possibility that the gene disrupted in suv2 is involved in a pathway that regulates cell-cycle progression when DNA is damaged or when replication is disturbed By contrast, GUS expression after UVB treatments was not significantly different among wild-type, suv2-1 and atr5 (Figure 6c), which is inconsistent with the severe root growth inhibition of suv2-1 and atr-5 by UVB irradiation The relatively weak GUS staining in wild-type roots could ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 Novel checkpoint gene in Arabidopsis 513 (a) (b) Figure Structure of the MRA19.1 gene and comparison of the MRA19.1 protein with proteins of the ATRIP/Rad26 family (a) Structure of the MRA19.1 gene, predicted ORF and insertions in suv2-1 and suv2-2 The MRA19.1 gene consists of 11 originally annotated exons (open boxes) and one newly identified exon (exon 2, filled box), which encodes a 646 amino acid protein (bottom) The region predicted to form a coiled-coil is shaded The stars indicates putative target sites of PIKK The suv2-1 mutant has an insertion of a 2110 bp fragment in intron 7, while suv2-2 has a T-DNA insertion in exon 10 (b) Comparison of the predicted MRA191 protein with ATRIP/Rad26 family proteins The structures of Homo sapience (human) ATRIP (NP_569055), Drosophila melanogaster (fly) MUS304 (NP_524135), Saccharomyces cerevisiae (budding yeast) DDC2 (LCD1; NP_010787) and Schizosaccharomyces pombe (fission yeast) Rad26 (CAA54058) are compared with that of MRA19.1 The region predicted to form a coiled-coil is shaded The stars indicate the positions of SQ/TQ motifs Figure UVB sensitivities of wild-type, suv2 and suv2 carrying the 35S:MRA19.1 gene Three-day-old seedlings were irradiated with kJ m)2 of UVB and grown for another days under continuous white light The root growth after the irradiation was measured using NIH Image Each value is the mean Ỉ SD of 8– 20 measurements be due to cell-cycle arrest that occurs before G2 (i.e in S phase) The MRA19.1 gene product dimerizes through the coiled-coil domain The presence of a coiled-coil domain in the MRA19.1 protein suggested that this protein might form a multimer Figure HU sensitivities of wild-type, atr, suv2 and the atr suv2 double mutant Three-day-old seedlings were transplanted onto new plates supplemented with 0.125–0.5 mM HU The seedlings were grown for another days under continuous white light The root growth after transplantation was measured using NIH Image, and expressed as a percentage of the mean length of wildtype roots on the non-supplemented plates Each value is the mean Ỉ SD of 18–20 measurements inside the cell To examine this possibility, we used a yeast two-hybrid system A full-length or partial MRA19.1 cDNA was sub-cloned into two types of destination vector (pDEST22 or pDEST32) to express the cDNA as fusion proteins with a DNA-binding domain (BD) or activation domain (AD) When the full-length sequence of MRA19.1 cDNA was inserted into both plasmids, yeast cells harboring both plasmids (BD-F and AD-F) could survive on the selection medium, suggesting the presence of a protein–protein interaction between the MRA19.1 proteins (Figure 7) When the N-terminal part of MRA19.1 covering the coiled-coil region was fused with AD (AD-NT) and combined with BD-F, yeast cells harboring both plasmids also survived on the selection medium, although growth was slower compared to the cells carrying BD-F and AD-F However, the yeast cells carrying AD-NT2 or AD-C, which lacked the coiled-coil domain, together with BD-F could not survive on the selection medium (Figure 7) In addition, the combination of BD-NT and AD-NT also allowed the yeast to survive on the selection medium (data not shown) These results suggest that the MRA19.1 protein forms multimers in yeast cells through the coiled-coil domain However, the more vigorous growth of yeast cells carrying BD-F and AD-F than those carrying BD-F and AD-NT suggests that domains other than the coiled-coil domain may contribute to stronger subunit interactions (Figure 7) AtATRIP is a member of ATRIP/Rad26 protein family Human ATRIP has been identified as an ATR-interacting protein that co-localizes with ATR in intra-nuclear foci ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 514 Ayako N Sakamoto et al (a) Figure Dimer formation of MRA19.1 proteins in yeast cells Full-length (F) MRA19.1 cDNA was used to prepare a bait plasmid Full-length (F) MRA19.1 cDNA, the N-terminal region containing the putative coiled-coil region (NT), the C-terminal region (CT) and the N-terminal region without the putative coiled-coil region (NT2) were used to prepare prey plasmids Combinations of bait and prey plasmids were introduced into yeast cells to express fusion proteins At least three independent clones were chosen randomly and grown on non-selection medium (NSM) or selection medium (SM) to examine the interaction between the fusion proteins (b) (c) Figure Expression of CYCB1;1:GUS after c-ray, UVB or aphidicolin treatment Wild-type (Columbia), atr or suv2 seedlings carrying the CYCB1;1:GUS gene were either exposed to UVB or c-rays, or placed in liquid medium containing aphidicolin After treatment and growth, the seedlings were fixed with 90% acetone and stained in X-Gluc solution (a) Expression of CYCB1;1:GUS after c-ray exposure Irradiated (100 Gy) or mock-irradiated (0 Gy) seedlings were grown under continuous white light for the indicated period, then fixed and stained (b) Expression of CYCB1;1:GUS after aphidicolin treatment Seedlings were placed in liquid medium supplemented with 0.1% DMSO (0 lg ml)1) or 12 lg ml)1 aphidicolin dissolved in DMSO (12 lg ml)1) for 24 h, then fixed and stained (c) Expression of CYCB1;1:GUS after UVB exposure Irradiated (2 kJ m)2) or mock-irradiated (0 kJ m)2) seedlings were grown under continuous white light for h, then fixed and stained following DNA damage or inhibition of replication (Cortez et al., 2001) Reducing ATRIP and consequently ATR levels by the use of small interfering RNAs resulted in the loss of a checkpoint response to DNA damage, suggesting that ATRIP and ATR are mutually dependent partners in cell-cycle checkpoint signaling pathways (Cortez et al., 2001) Mec1 and Rad3 are homologues of ATR in Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively (Bentley et al., 1996), and their respective partners, Ddc2 and Rad26, are also thought to be homologues of ATRIP (Figure 3b) ATRIP dimerizes through the coiled-coil motif (Itakura et al., 2005), and interacts with ATR with its N-terminal region (Itakura et al., 2004a) ATRIP is phosphorylated in an ATRdependent manner (Itakura et al., 2004b) Likewise, Rad26 or Ddc2 are phosphorylated in a Rad3- or Mec1-dependent manner, respectively (Edward et al., 1999; Paciotti et al., 2000) The serine residues in the conserved TQ/SQ sequence are targets for direct modification by ATR in vitro (Itakura et al., 2004b) Activated ATR phosphorylates a number of proteins, including mediators or effector kinases, through the TQ/SQ sequences (Bao et al., 2001; Zhao and PiwnicaWorms, 2001), which transmits the checkpoint signal to the cell-cycle regulators The phenotype of the suv2 mutant suggests a close functional interaction with AtATR and involvement in the checkpoint pathway The suv2 mutant showed reduction of G2/M arrest after c-ray exposure or aphidicolin treatment, similar to atr mutants Both suv2 and atr single mutants and the double mutant displayed indistinguishable sensitivity to UVB and HU Moreover, MRA19.1 (SUV2) proteins dimerize in yeast cells through the coiled-coil domains The amino acid sequence of MRA19.1 has two instances of the SQ sequence that is a potential target of PIKKs, including ATR The amino acid sequence surrounding the two SQs is well conserved among plant cell-cycle-related genes (Table 1), suggesting that this motif is functional in the signal transduction pathway in plants These lines of evidence strongly suggest that the protein encoded by MRA19.1 is a member of the ATRIP/Rad26 family Thus, we renamed this gene AtATRIP Root growth arrest and checkpoint response After exposure to the DNA-damaging agents or replication inhibitors, the root growth rate of wild-type plants was slightly reduced compared to that of non-treated plants (Figures and 5) In agreement with this, abundant accumulation of CYCB1;1:GUS in c-irradiated or aphidicolin-treated wild-type roots was observed, suggesting that a correct checkpoint response occurred in the meristematic tissues (Figure 6a,b) Thus, reduced root growth in the wild-type root is thought to be due to temporal arrest of the cell cycle by checkpoint responses Similarly, the root growth of atr and suv2 plants treated with the ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 Novel checkpoint gene in Arabidopsis 515 Table Conserved SQ/TQ sequence in plant cell-cycle-related proteins Protein MRA19.1 Rice hypothetical protein (Indica) Rice hypothetical protein (Japonica) AtWEE1 AtATR AtRA17 AtRAD9 AtNBS1 AtREV1 Position of S or T Sequence Reference 87 152 30 300 452 522 91 111 381 533 603 15 95 190 484 765 790 1032 1183 1789 1923 2043 2079 2588 54 246 273 431 34 203 266 367 371 389 210 275, 278 442 180 909 1072 SPPRELSQRVVSGF LKEQECSQLKKGKS SPPRELSQRPAAEV SVRNAISQVYDIII RCENDRSQEAISSI IMEVLASQMEYDTA LPPRELSQHPQGFD SPPRELSQRPAASP SVRNAISQVYDIII RCENDRSQEAISSI IMEVLASQMEYDTA LLAKRKTQGTIKIR EKDFILSQDFFCTP KRLAARSQLFLQVY PAGPSLSQTESSIV DESSEETQLACVEV ILVRTRSQWIRCLQ VLQLYHSQIGSDNQ SIKYVISQIFAALI YKKTWCTQGVQAAW ENRLRFTQSSLWTR EPTVRNTQSFKEKK DVLNLYTQVKELLP LVPFRLTQNMIDGL LSGASATQEDSSLV LLLVKSTQIPTVIL ARRMEDTQSSLERA APEKVLSQAHGQAG LVQASPTQLALHTL GDSLLHTQLWIDPS LATMLVSQLQEGIP PAAPPKSQRPTQID PKSQRPTQIDHAEG VQNQSFSQHHPSNW SEIPGYSQYRPSVM EDISSMSQDSQFGEINR GSEIIYTQDLIVRD LDQLNDTQPKLSAF LLPSSLSQVDVSVL KRLAARSQLFLQVY This study EEC72403 EAZ21598 De Schutter et al (2007) Culligan et al (2004) Heitzeberg et al (2004) Heitzeberg et al (2004) Waterworth et al (2007) Takahashi et al (2005) DNA-damaging agents or replication inhibitors was also inhibited compared to non-treated plants (Figures and 5) In these mutants, CYCB1;1:GUS accumulation, induced by c-irradiation or aphidicolin, was less than that in wildtype plants (Figure 6a,b) At first glance, this may seem consistent with the view that disruption of ATR or AtATRIP allows the root cells to grow without cell-cycle arrest However, this is a paradoxical situation because the root growth was actually more severely inhibited by DNAdamaging agents or replication inhibitors in those mutants than in wild-type There are several possible explanations for this One possibility is that the some cells fail to arrest at G2/M but instead are arrested at G1/S in the atr and suv2 plants It has been suggested that HU treatment induces G1 arrest in addition to G2 arrest in atr mutant plants (Culligan et al., 2004) It is possible that the c-irradiated atr and suv2 plants may be arrested at G1 phase, and recovery from this arrest may be delayed Another possibility is that the cells that failed to arrest at G2/M accumulate DNA damage and/or chromosome defects, resulting in inactivated cells (which exit the cell cycle) or cell death Culligan et al (2004) reported that cell death occurs in aphidicolin-treated atr plants In addition, Curtis and Hays (2007) detected inactivated cells in UVB-irradiated wild-type roots by propidium iodide staining These lines of evidence support the view that cell inactivation or death may cause root growth inhibition in atr and suv2 plants Although the root growth of wild-type plants was inhibited by irradiation with kJ m)2 of UVB (Figure 1a,b), induction of CYCB1;1:GUS accumulation by UV- rradiation was much weaker than that caused by c-irradiation or aphidicolin treatment (Figure 1c and data not shown) This could be because the UV-irradiated cells are arrested at a different phase to G2/M It is known that UV irradiation arrests the cell cycle at various stages in other organisms (Siede et al., 1994; Orren et al., 1995; Heffernan et al., 2002) Thus, UVB treatment may bring about cell-cycle arrest at G1 or S phase rather than, or in addition to, G2 phase in wildtype plants, although it is still unclear why more severe growth inhibition was seen in the atr and suv2 roots versus wild-type plants Further analysis with various cell-cycle markers is required to evaluate the mechanism of cell-cycle checkpoints in higher plants EXPERIMENTAL PROCEDURES Plant materials and mutant isolation The wild-type plant used in this study was Arabidopsis thaliana Columbia (Col) The suv2-1 mutant (Col) was isolated from the carbon ion-irradiated M2 seed stocks described previously (Sakamoto et al., 2003) The suv2-2 (SALK_077978) and atr-5 (SALK_083543) mutants were provided by Arabidopsis Biological Resource Center Mapping analysis and segregation test The suv2-1 plant was crossed with the Landsberg erecta (Ler) ecotype to obtain an F2 genetic mapping population F2 plants from this cross were grown, and DNA was extracted from individual plants for genetic marker analysis The individual plants were then allowed to self-pollinate to obtain F3 seeds (F2 line seed pool) Approximately ten F3 seedlings from each F2 line were examined for UVB sensitivity using a root-bending assay (Britt et al., 1993) F2 lines for which all seedlings displayed a UVB-hypersensitive phenotype (and therefore homozygous for suv2-1) were used to establish the genetic mapping population of 304 lines To determine the genetic map location of suv2-1, this mapping population was analyzed using SSLP (Bell and Ecker, 1994) or CAPS (Konieczny and Ausubel, 1993) genetic markers The sequences of these PCR-based markers ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 516 Ayako N Sakamoto et al are listed in the TAIR database (http://www.Arabidopsis.org/) or in Table S1 The suv2-1 plant was back-crossed with the parent Columbia, and approximately 100 F2 plants derived from this cross were grown and self-pollinated to produce F2 lines More than 20 seedlings per F2 line were examined for UVB sensitivity, and the lines in which all seedlings showed UV sensitivity were identified as homozygous UV-sensitive lines The number of UV-sensitive lines and other lines was examined by v2 test Isolation of MRA19.1 cDNA and complementation test To isolate the full-length MRA19.1 cDNA, total RNA was extracted from wild-type plants and used in RT-PCR reactions with primers RT-F1 (5¢-AACCAAGGTCTTCAAATTTACA-3¢) and RT-R1 (5¢-TACTATGCTCCTTCAATCAAAAT-3¢) The RT-PCR products were subcloned into pGEM-T vectors (Promega, http://www.promega.com/) and sequenced The original full-length cDNA contained four PCR errors, which were removed by digesting the fragment with restriction enzymes and replacing them with independently amplified short PCR products To transfer the MRA19.1 cDNA into the entry vector, the cDNA was re-amplified using primers 5¢-CACCATGGCGAAGGACGACAATAA-3¢ and 5¢-TCATATAGTATTATCACC-3¢, and transferred into the pENTR/D-TOPO vector (Invitrogen, http://www.invitrogen.com/) For the complementation test, the inserts were transferred into binary vector pB7WG2 (Karimi et al., 2002), and used to transform Agobacterium strain GV3101 The suv2-2 plants were infected with pB7WG2-transformed Agrobacterium, and T1 plants were selected on selection medium (1 · MS, · B5 vitamin mix, 2.5% sucrose, 0.2% gellan gum, 0.02 mg ml)1 of glufosinate ammonium, pH 6.3) Coiled-coil region prediction and helical wheel analysis Prediction of coiled-coil regions was performed at http://www ch.embnet.org/software/COILS_form.html (Lupas et al., 1991) Helical wheel analysis was done according to the Schiffer-Edmundson wheel model (Schiffer-Edmundson, 1967) Analysis of plant sensitivities to UVB, c-rays, MMC and HU damage or cell-cycle inhibition, 3-day-old seedlings were exposed to UVB or c-rays or put into liquid medium (2% sucrose, 0.1% v/v commercial nutrient) containing 12 lg ml)1 aphidicolin After the indicated period, the plants were fixed with 90% acetone for 30 min, washed with 0.1 M sodium phosphate buffer (pH 7.2), and soaked overnight in X-Gluc solution [0.1 M sodium phosphate buffer (pH 7.2) with 0.1% Triton X-100, 0.05% sodium azide, 0.5 mM potassium hexacyanoferrate (III), 0.5 mM potassium hexacyanoferrate (II) trihydrate and 0.95 mM 5-bromo-4-chloro-3-indoxyl-b-D-glucuronic acid cyclohexylammonium salt) Seedlings were then washed in 70% ethanol overnight to clear the tissue, and GUS expression in the root tip was observed under light microscopy Yeast two-hybrid assay Dimer formation of MRA19.1 was examined using the Pro-Quest two-hybrid system (Invitrogen) Full-length or partial-length MRA19.1 cDNAs were amplified using PfuTurbo DNA polymerase (Agilent Technologies, http://www.home.agilent.com) and cloned into pENTR/D-TOPO or pENTR/SD/D-TOPO (Invitrogen) The inserts were then transferred to pDEST22 or pDEST32 using Gateway LR Clonase II (Invitrogen) to prepare bait and prey plasmids Yeast strain MaV203 was transformed with a pairwise combination of bait and prey plasmids, and screened on SC-Leu-Trp medium according to the instructions in the ProQuest Two-hybrid System (Invitrogen) To detect the interaction between two fusion proteins, the yeast clone harbouring two plasmids were grown in liquid medium for 20 h, and the cells were harvested and resuspended in water Yeast cell suspension (5 ll at an absorbance at 600 nm of approximately 0.2) was dropped on selection medium (SC-Leu-Trp-His supplemented with 25–50 mM 3-aminotriazole) or non-selection medium (SC-Leu-Trp), and incubated at 30°C for days ACKNOWLEDGEMENTS We thank Anne B Britt (University of California, Davis) and Kevin M Culligan (University of New Hampshire) for critical reading of this manuscript We also thank Chihiro Suzuki and Akari Nakasone for their technical assistance, and Yutaka Oono for helpful comments We acknowledge Peter Doerner (University of Edinburgh) for providing CYCB1;1:GUS transgenic plants, the Arabidopsis Biological Resources Center for providing the Arabidopsis T-DNA insertion mutants and BAC clones, and Kazusa DNA Research Institute for providing the P1 clones MRA19 and MCL19 This work was partially supported by a grant from the Japan Society for Promotion of Science (19570049) to A.N.S Seeds were set on nutritive agar plates (2% sucrose, 1.5% agar, 0.1% v/v commercial nutrient Hyponex (Hyponex Corporation, http://www.hyponex.co.jp/link/index.html) and grown vertically under continuous white light (approximately 40 mE m)2 s)1) for days at 23°C For analysis of UVB sensitivity, seedlings were exposed to UVB from a UV lamp (CSL-30B; COSMO BIO Co Ltd, http://www.cosmobio.co.jp/index_e.asp) at a dose rate of approximately 0.25 kJ m)2 min)1 To test c-ray sensitivity, 3-day-old seedlings were irradiated with c-rays from a 60Co irradiation facility (Japan Atomic Energy Agency, http://www.jaea.go.jp/english/ index.shtml) with a dose rate of 100 Gy h)1 To test MMC or HU sensitivity, 3-day-old seedlings were transplanted to the surface of nutritive agar plates supplemented with MMC or HU After irradiation or transplantation, the plates were immediately positioned vertically so that the new root growth was at right angles to the previous growth The plants were then incubated in the dark or under continuous white light for days at 23°C, and new root growth was measured using NIH Image version 1.62 (http://rsb info.nih.gov/nih-image/) Additional Supporting Information may be found in the online version of this article: Figure S1 Detection of the insertion in the suv2-1 mutant Figure S2 Structure of the AtATR gene and semi-quantitative RT-PCR analysis of AtATR and MRA19.1 Table S1 Nucleotide sequences for PCR primers Table S2 PCR primers to detect chromosome rearrangement Table S3 Specific primer for TAIL-PCR Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article Expression of CYCB1;1:GUS REFERENCES The CYCB1;1:GUS construct was introgressed to the atr-5 or suv2-1 background by genetic crossing each mutant to a CYCB1;1:GUScontaining Columbia line To detect GUS expression after DNA Ahmad, M., 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and Culligan, K.M (2009) The Arabidopsis ATRIP ortholog is required for programmed response to replication inhibitors Plant J doi: 10.1111/j.1365-313X.2009.03975.x Takahashi, S., Sakamoto, A., Sato, S., Kato, T., Tabata, S and Tanaka, A (2005) Roles of Arabidopsis AtREV1 and AtREV7 in translesion synthesis Plant Physiol 138, 870–881 Waterworth, W.M., Altun, C., Armstrong, S.J., Roberts, N., Dean, P.J., Young, K., Weil, C.F., Bray, C.M and West, C.E (2007) NBS1 is involved in DNA repair and plays a synergistic role with ATM in mediating meiotic homologous recombination Plant J 52, 41–52 Zhao, H and Piwnica-Worms, H (2001) ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1 Mol Cell Biol 21, 4129–4139 The Genbank accession number for the AtATRIP mRNA is AB492859 ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 509–517 ... regulated by ATR is transferred to the cyclin/CDK complex (through the ATR activity) in Arabidopsis (Culligan et al., 2006) In Arabidopsis, a downstream target of AtATM kinase, AtNBS1, has been... is reminiscent of the atr mutant The characteristics of the MRA19.1 protein suggests that this gene may be involved in cell-cycle checkpoint regulation and possibly associated with AtATR in Arabidopsis. .. Arabidopsis To compare the phenotype of AtATR-disrupted plants with that of suv2, we obtained a T -DNA insertion mutant of ATR (atr-5; SALK_083543) Semi-quantitative RT-PCR analysis revealed that