The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis
2016; Springer Nature; Volume: 35; Issue: 19 Linguagem: Inglês
10.15252/embj.201593083
ISSN1460-2075
AutoresAnnika K. Weimer, Sascha Biedermann, Hirofumi Harashima, Farshad Roodbarkelari, Naoki Takahashi, Julia Foreman, Yonsheng Guan, Gaëtan Pochon, Maren Heese, Daniël Van Damme, Keiko Sugimoto, Csaba Koncz, Peter Doerner, Masaaki Umeda, Arp Schnittger,
Tópico(s)Plant Genetic and Mutation Studies
ResumoArticle6 August 2016Open Access Transparent process The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis Annika K Weimer Annika K Weimer Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Sascha Biedermann Sascha Biedermann Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Hirofumi Harashima Hirofumi Harashima orcid.org/0000-0003-3370-4111 RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, Japan Search for more papers by this author Farshad Roodbarkelari Farshad Roodbarkelari Institut für Biologie III, Universität Freiburg, Freiburg, Germany Search for more papers by this author Naoki Takahashi Naoki Takahashi Plant Growth Regulation Laboratory, Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan Search for more papers by this author Julia Foreman Julia Foreman School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yonsheng Guan Yonsheng Guan Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Gaëtan Pochon Gaëtan Pochon Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Search for more papers by this author Maren Heese Maren Heese orcid.org/0000-0002-1070-9107 Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Search for more papers by this author Daniël Van Damme Daniël Van Damme Department of Plant Systems Biology, VIB, Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium Search for more papers by this author Keiko Sugimoto Keiko Sugimoto RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, Japan Search for more papers by this author Csaba Koncz Csaba Koncz Max-Planck-Institut für Pflanzenzüchtungsforschung, Köln, Germany Search for more papers by this author Peter Doerner Peter Doerner School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Masaaki Umeda Masaaki Umeda Plant Growth Regulation Laboratory, Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan JST, CREST, Ikoma, Nara, Japan Search for more papers by this author Arp Schnittger Corresponding Author Arp Schnittger [email protected] orcid.org/0000-0001-7067-0091 Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Trinationales Institut für Pflanzenforschung, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS, Strasbourg Cedex, France Search for more papers by this author Annika K Weimer Annika K Weimer Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Sascha Biedermann Sascha Biedermann Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Hirofumi Harashima Hirofumi Harashima orcid.org/0000-0003-3370-4111 RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, Japan Search for more papers by this author Farshad Roodbarkelari Farshad Roodbarkelari Institut für Biologie III, Universität Freiburg, Freiburg, Germany Search for more papers by this author Naoki Takahashi Naoki Takahashi Plant Growth Regulation Laboratory, Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan Search for more papers by this author Julia Foreman Julia Foreman School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yonsheng Guan Yonsheng Guan Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Search for more papers by this author Gaëtan Pochon Gaëtan Pochon Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Search for more papers by this author Maren Heese Maren Heese orcid.org/0000-0002-1070-9107 Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Search for more papers by this author Daniël Van Damme Daniël Van Damme Department of Plant Systems Biology, VIB, Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium Search for more papers by this author Keiko Sugimoto Keiko Sugimoto RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, Japan Search for more papers by this author Csaba Koncz Csaba Koncz Max-Planck-Institut für Pflanzenzüchtungsforschung, Köln, Germany Search for more papers by this author Peter Doerner Peter Doerner School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Masaaki Umeda Masaaki Umeda Plant Growth Regulation Laboratory, Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan JST, CREST, Ikoma, Nara, Japan Search for more papers by this author Arp Schnittger Corresponding Author Arp Schnittger [email protected] orcid.org/0000-0001-7067-0091 Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany Trinationales Institut für Pflanzenforschung, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS, Strasbourg Cedex, France Search for more papers by this author Author Information Annika K Weimer1,12, Sascha Biedermann1, Hirofumi Harashima2, Farshad Roodbarkelari3, Naoki Takahashi4, Julia Foreman5, Yonsheng Guan1, Gaëtan Pochon6, Maren Heese6, Daniël Van Damme7,8, Keiko Sugimoto2, Csaba Koncz9, Peter Doerner5, Masaaki Umeda4,10 and Arp Schnittger *,1,6,11 1Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS UPR2357, Université de Strasbourg, Strasbourg Cedex, France 2RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, Japan 3Institut für Biologie III, Universität Freiburg, Freiburg, Germany 4Plant Growth Regulation Laboratory, Nara Institute of Science and Technology, Graduate School of Biological Sciences, Ikoma, Nara, Japan 5School of Biological Sciences, University of Edinburgh, Edinburgh, UK 6Department of Developmental Biology, Biozentrum Klein Flottbek, University of Hamburg, Hamburg, Germany 7Department of Plant Systems Biology, VIB, Ghent, Belgium 8Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium 9Max-Planck-Institut für Pflanzenzüchtungsforschung, Köln, Germany 10JST, CREST, Ikoma, Nara, Japan 11Trinationales Institut für Pflanzenforschung, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS, Strasbourg Cedex, France 12Present address: Department of Biology, Stanford University, Stanford, CA, USA *Corresponding author. Tel: +49 40 428 16 502; Fax: +49 40 428 16 503; E-mail: [email protected] The EMBO Journal (2016)35:2068-2086https://doi.org/10.15252/embj.201593083 See also: B Desvoyes & C Gutierrez (October 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR. Synopsis Cell division suppression upon DNA damage depends on inhibition of mitotic cyclin-dependent kinase (CDK) activity, yet CDKs also promote DNA repair functions. Arabidopsis solves this problem by utilizing an alternative, plant-specific CDKB1-CYCB1 complex to stimulate homologous recombination repair. Plant-specific B1-type CDKs (CDKB1s) and B1-type cyclins (CYCB1s) regulate homologous recombination. CDKB1 and CYCB1 are required for lesion localization of the key recombinase RAD51. RAD51 is a substrate of CDKB1-CYCB1 complexes. The p53 functional homolog SOG1 controls CDKB1-CYCB1 activation upon DNA damage. Introduction DNA damage is a crucial problem for every organism and many repair pathways exist to recover from the different types of DNA damage. Of key importance after DNA damage is an arrest of cell division to allow sufficient time for repair and to prevent that mutated daughter cells are generated that will propagate incorrect genetic information. One severe type of DNA damage often caused by irradiation or chemical mutagens is double-strand breaks (DSBs), and the signaling cascades from DSBs to cell division arrest are well understood in yeast and animals. In essence, DSBs induce the activity of the kinase ataxia-telangiectasia mutated (ATM) that phosphorylates and activates checkpoint kinase 2 (Chk2). Chk2 in turn inhibits the Cdc25 phosphatase, a central activator of the main cell-cycle regulators Cdk1 and Cdk2 in animals. In addition, the ATM pathway activates Wee1, a negative regulator of Cdk1 and Cdk2 providing a parallel block of the cell cycle (Kastan & Bartek, 2004; Harper & Elledge, 2007; Yata & Esashi, 2009). Remarkably, plants can cope with very high concentrations of harmful agents in comparison with animals. For instance, a comparative study of tobacco BY-2 and Chinese hamster ovary cells showed that plant cells yielded one-third less double-strand breaks after the same dose of ionizing radiation (IR). Furthermore, the plant cells also tolerated a much higher number of DSBs before they died (Yokota et al, 2005). Despite the apparent power and their relevance for agriculture under changing environmental conditions, the plant DNA repair pathways are not very well understood. Moreover, the canonical response pathways of yeast and animals appear to be only partially conserved. While homologs of ATM and its sister kinase ATR (ATM- and Rad3-related) predominantly involved in replication stress response by sensing single-stranded DNA have also been identified in Arabidopsis (Garcia et al, 2000; Culligan et al, 2004; Culligan & Britt, 2008), no homologs of Chk2 or its sister kinase Chk1 could be found in plants to date. Furthermore, even though a homolog of the yeast Wee1 kinase exists in Arabidopsis and other plants, its function appears to be different as Arabidopsis WEE1 was found to act during S phase after hydroxyurea (HU)-induced replication stress and not in repressing CDK activity during mitosis or blocking cell division after DSB formation (De Schutter et al, 2007; Cools et al, 2011). Moreover, transgenic plants expressing a mutant version of CDKA;1, the Arabidopsis homolog of mammalian Cdk1 and Cdk2, in which the putative WEE1 target sites were replaced with non-phosphorylatable amino acids, were not hypersensitive to HU indicated that cell-cycle arrest after DNA damage is differently regulated in plants (Dissmeyer et al, 2009, 2010). Besides CDKA;1, plants contain B-type CDKs that have been implicated in cell-cycle control. While there appears to be only a single B-type CDK in the unicellular algae Chlamydomonas rheinhardii that is essential for mitosis (Bisova et al, 2005; Tulin & Cross, 2014), B-type CDKs are divided into a B1 and B2 class in Arabidopsis and other multicellular plants. B2-type CDKs appear to be major regulators of mitosis in Arabidopsis and their loss as well as their overexpression interferes with cell proliferation hinting at a strong dose-dependent action (Andersen et al, 2008). However, due to the lack of mutants, a detailed analysis of B2-type kinases is still pending. In contrast, B1-type CDKs have been functionally analyzed, but these studies revealed so far that they apparently act as axillary kinases to A1-type kinases contributing to the refinement of developmental decisions (Xie et al, 2010; Cruz-Ramirez et al, 2012; Nowack et al, 2012; Weimer et al, 2012). Another obvious difference between plants and other well-studied eukaryotes is the presence of a large groups of cyclins, for example, more than 30 cyclins in Arabidopsis, most of which are still uncharacterized (Harashima et al, 2013). Very little is known about the regulation of these cyclins but remarkably, previous studies have revealed that CYCB1;1 is upregulated during various treatments of DNA damage-inducing agents or in mutants affected in chromatin organization, DNA metabolism, and/or repair such as fasciata 1 (fas1), jing he sheng 1 (jhs1), and dna replication factor c1 (rfc1) (Chen et al, 2003; Culligan et al, 2004; Endo et al, 2006; Liu et al, 2010; Adachi et al, 2011; Jia et al, 2016). This upregulation is remarkable due to the predicted role of these cyclins in promoting cell division. Up to now, it was not clear what the role of B1-type cyclins in DNA damage response is, especially in which DNA damage pathway they could act. In plants as well as in other organisms, two major DNA repair pathways are responsible for genomic integrity after DNA double-strand breaks: non-homologous end-joining (NHEJ) and homology-dependent repair, also called homologous recombination repair (HR). With NHEJ, the damaged DNA is repaired by direct ligation of the broken ends. The double-strand break is recognized by a KU70/KU80 heterodimer and then processed by the MRN complex that is composed of MRE11 (MEIOTIC RECOMBINATION 11), RAD50 (RADIATION SENSITIVE 50), and NBS1 (NIJMEGEN-BREAKAGE SYNDROME 1) (Amiard et al, 2013). DNA ends are ligated by LIG4 (DNA LIGASE 4) and XRCC4 (X-RAY REPAIR CROSS-COMPLEMENTATION PROTEIN 4) (Bray & West, 2005). Consistently, ku70 and ku80 mutants are hypersensitive to the DSB-inducing agents bleomycin (BLM) and methyl methane sulfonate (MMS) (Riha et al, 2002). However, NHEJ can be imprecise, leading to the loss of nucleotides when overlaps are not compatible (Takata et al, 1998). In contrast to NHEJ, HR is highly accurate since it exactly replaces the defective DNA (Shrivastav et al, 2008). HR requires a homologous template to repair the damaged DNA and can therefore only occur after DNA replication in S phase and the subsequent G2 phase of the cell cycle when sister chromatids are available. This pathway is initiated by the resection of DNA, also executed by the MRN complex, and formation of long 3′ tails, which are coated by RPA (REPLICATION PROTEIN A) in order to prevent winding of the DNA. Homology search and strand invasion are performed by RAD51 family members (Serra et al, 2013), the eukaryotic homolog of the E. coli recA protein (Mengiste & Paszkowski, 1999). RAD51 has five paralogs in Arabidopsis (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), all of which function in HR in somatic or meiotic cells and show fewer homologous recombination events after DNA damage (Abe et al, 2005; Da Ines et al, 2013a,b; Serra et al, 2013), which is in line with studies in other eukaryotes (Hosoya & Miyagawa, 2014). A key question is how cells can decide whether to follow NHEJ or enter an HR pathway. While this decision appears to be complex and likely also involves developmental factors, many studies have revealed that CDKs play an important role in the choice of the repair pathway based on the observation that mitotic CDK activity is rising after S phase and hence allowing a cell to discriminate between a G1 and a G2 phase (Wohlbold & Fisher, 2009; Yata & Esashi, 2009; Trovesi et al, 2013). Moreover, CDKs were found to be directly involved in promoting HR. However, the requirement of active CDKs for HR causes an apparent dilemma for a cell since mitotic CDK activity needs to be shut down to arrest the cell division program as a first measure to DNA damage. Here, we show that plants solve this problem by specifically activating B1-type CDKs at a transcriptional and posttranslational level after DNA damage. With this, we reveal a previously not recognized key function of B1-type CDKs as central regulators of DNA damage response in plants. We show that CYCB1s are the specific partner of CDKB1 during DNA damage and both form active complexes that can phosphorylate RAD51. Moreover, we show that HR and NHEJ pathways act at least partially redundantly on DSB, possibly contributing to the powerful DNA damage repair system of plants. Results Mutants for B1-type cyclins are specifically hypersensitive to DNA cross-links Based on the observation that CYCB1;1 is upregulated during treatments with DNA damage-inducing agents (Chen et al, 2003; Culligan et al, 2004, 2006; Ricaud et al, 2007; Adachi et al, 2011), we isolated mutants in all four B1-type cyclins to address a possible role of these cyclins in DNA stress (Fig EV1A–C). To this end, we monitored root growth of Arabidopsis plants on agar plates containing different DNA-damaging drugs (see below). On control plates without DNA damage agents, none of the cycb1 mutants showed altered root growth in comparison with the wild type (Figs 1A and B, and EV2A–D and I). Click here to expand this figure. Figure EV1. T-DNA insertion mutants of the Arabidopsis thaliana CYCB1 genes Schematic overview of T-DNA insertions for cycb1;1, cycb1;2, cycb1;3, and cycb1;4. Black boxes display exons, and red arrowheads indicate the positions of the T-DNA insertion. Quantitative PCR for relative expression levels in the wild type (Col-0), cycb1;1, cycb1;2 and cycb1;4. EXP, SAND, and ACT7 were used as reference genes for normalization. Each value represents the mean ± standard deviation of three independent experiments. Quantitative PCR for relative expression levels in the wild type (Nos-0) and cycb1;3. EXP, SAND, and ACT7 were used as reference genes for normalization. Each value represents the mean ± standard deviation of three independent experiments. Download figure Download PowerPoint Figure 1. Mutants of B1-type cyclins are hypersensitive to cisplatin A. cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4, and the wild type (from left to right) on control plates without genotoxic agent 10 days after germination. B. The wild type, single, and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination. C. The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 1 mM hydroxyurea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU. D. The wild type, single, and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination. E. The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 0.6 μg/ml bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM. F. The wild type, single, and double mutants of cycb1 were grown on plates supplemented with 0.6 μg/ml BLM. Root lengths were measured 10 days after germination. G. The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 15 μM cisplatin 6 days after germination, that is, 3 days after transfer from control plates. H–J. cycb1 mutants were germinated on control plates and were transferred to new control plates (H) or plates supplemented with 15 μM (I) or 30 μM (J) cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs. K. The wild type, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 (from left to right) on plates containing 30 μM cisplatin 6 days after germination, that is, 3 days after transfer from control plates. L–N. cycb1 double mutants germinated on control plates and were transferred to new control plates (L) or plates supplemented with 15 μM (M) or 30 μM (N) cisplatin 3 days after germination. Root lengths were measured 3 days after transfer and the net root growth of 3 days is shown in the graphs. Data information: One or two asterisks indicate significant differences within a 5 and 1% confidence interval, respectively (Student's t-test). Scale bars: 1 cm. Three biological replicates, each containing at least 15 plants, were analyzed. The mean of the root length of each individual experiment was determined and again averaged for the three biological replicates. Graphs represent mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Phenotype of cycb1 mutants under greenhouse conditions A–D. Single mutants cycb1;1 (A), cycb1;2 (B), cycb1;3 (C), and cycb1;4 (D). E–G. Double mutants cycb1;1 cycb1;2 (E), cycb1;1 ku70 (F), and cycb1;2 ku70 (G). Seedlings of cycb1;1 cycb1;2 are smaller and more diverse than wild-type plants. H. The triple mutant cycb1;1 cycb1;2 ku70 displays a similar phenotype as the double mutant cycb1;1 cycb1;2. I. Wild type. J. Single mutant ku70. Data information: All images were taken 14 days after germination. Scale bars: 1 cm. Download figure Download PowerPoint First, we tested root growth on media containing HU, which causes intra-S-phase stress due to the inhibition of the enzyme ribonucleotide reductase and thus a decrease in production of deoxyribonucleotides (Yarbro, 1992). For this analysis, wee1 was used as a positive control and, consistent with previous data, was found to be highly sensitive to HU, whereas it shows no growth abnormalities on control medium (De Schutter et al, 2007; Cools et al, 2011). In contrast, root growth on HU of all tested cycb1 mutants was comparable to the growth of wild-type plants (Fig 1C and D). To address a possible redundant function among the CYCB1 group, we generated the double mutants cycb1;1 cycb1;2, cycb1;1 cycb1;3, cycb1;1 cycb1;4, cycb1;2 cycb1;4, and cycb1;3 cycb1;4. With the exception of cycb1;1 cycb1;2, all double mutants grew indistinguishably from the wild type on media with and without HU (Figs 1A–D and EV3A). The double mutant cycb1;1 cycb1;2 had shorter roots than the wild type on both media with and without DNA stress-inducing drugs (Fig 1A–D). Comparing the root growth ratios of plants grown on media without and with HU, it became obvious that this double mutant was not more sensitive than the wild type to HU (Fig EV3A). Click here to expand this figure. Figure EV3. Root growth ratios of cycb1 and cdkb1 mutants A, B. Graphs represent the ratio of the mean growth rate on 1 mM hydroxyurea (HU) and 0.6 μg/ml bleomycin (BLM) compared to control experiments on plates lacking genotoxins for the wild type and the double mutant cycb1;1 cycb1;2 (A) or the weak CDKA;1 allele DE (CDKA;1T15D;Y15E) (B). C. Graphs represent the ratio of the mean growth rate on 1 mM HU and 0.6 μg/ml BLM compared to control experiments on plates lacking genotoxins for the wild type, the single mutants cdkb1;1 and cdkb1;2, and the double mutant cdkb1;1 cdkb1;2. Download figure Download PowerPoint Next, root growth of single and double mutant combinations of cycb1s was tested on media containing the DSB-inducing drug BLM. As a positive control, we used mutants in ku70 that were shown to grow as the wild type on medium without drugs (Cools et al, 2011). Whereas ku70 mutants were sensitive to BLM and grew only very little consistent with previous reports (Tamura et al, 2002; West et al, 2002; Cools et al, 2011), no significant difference was found between cycb1 single and double mutants versus the wild type again with the exception of cycb1;1 cycb1;2 (Fig 1E and F). Comparing root growth ratios on plates with and without BLM indicated that cycb1;1 cycb1;2 is also not hypersensitive to this drug (Fig EV3A). As a third drug, the hypersensitivity of cycb1 mutants to cisplatin was tested. Cisplatin causes in addition to DNA breaks also intra- and interstrand DNA links that require repair by HR in contrast to damage caused by BLM and HU that can also be repaired by NHEJ (Kartalou & Essigmann, 2001; Belenkov et al, 2002; De Silva et al, 2002; Fuertes et al, 2002; Crul et al, 2003; Siddik, 2003; Pinato et al, 2014). Since cisplatin is unstable in solution, seedlings were germinated on media without the drug and then transferred to plates containing two concentrations of cisplatin (15 and 30 μM) 3 days after germination. On plates with 15 μM cisplatin, the net root growth of the cycb1 mutants at 3 days after the transfer appeared to be reduced but was not statistically significantly different from the growth of wild-type plants (Fig 1G–I, L and M). However, at 30 μM cisplatin, the roots of all B1-type cyclin mutants were significantly shorter than the roots of wild-type plants (Student's t-test P < 0.01) (Fig 1J and K). The observation that root growth of the cycb1 double mutants was not further reduced in comparison with the growth of the single mutants suggested that all four cyclins contribute in a non-additive manner to growth on media with cisplatin (Fig 1G, J, K and N). One exception was the double mutant cycb1;1 cycb1;4 that, while being shorter than the wild type, grew better than the other double mutants. However, since all single mutants including cycb1;1 and cycb1;4 as well as all other double mutant combinations are significantly shorter, we conclude that an indirect effect, for example, a compensatory action, in the cycb1;1 cycb1;4 double mutant triggers this response and that the general theme of mutants in B1-type cyclins is a hypersensitivity against cisplatin. Next, we asked whether the hypersensitivity of the cycb1 mutants and their reduced growth on cisplatin was due to increased cell death. To this end, we stained the wild type and cycb1 mutants with propidium iodide to visualize dying cells. Under control conditions, no cell death occurred in all tested genotypes. After cisplatin treatment, dead cells were observed in close proximity to the quiescent center. However, we did not see obvious difference between the wild type and cycb1 mutants indicating a higher level of cisplatin-induced DNA damage in the cycb1 mutants (Fig EV4). Click here to expand this figure. Figure EV4. Cell death in cycb1 mutants after cisplatin treatment A–L. Propidium iodide staining of the apical root meristem in wild type grown on control plates (A) or plates supplemented with 50 μM cisplatin for 24 h (B); cycb1;1 grown on control plates (C) or plates supplemented with 50 μM cisplatin for 24 h (D); cycb1;2 grown on control plates (E) or plates supplemented with 50 μM cisplatin for 24 h (F); cycb1;3 grown on control plates (G) or plates supplemented with 50 μM cisplatin for 24 h (H); cycb1;4 grown on control plates (I) or plates supplemented with 50 μM cisplatin for 24 h (J); cycb1;1 cycb1;2 grown on control plates (K) or plates supplemented with 50 μM cisplatin for 24 h (L). Red spots show cell death. Scale bars: 20 μm. Download figure Download PowerPoint Previously, it was reported that Arabidopsis root cells entered an endoreplication cycle in which the nuclear DNA is amplified without subsequent cell division as a response to zeocin-induced DNA damage (Adachi et al, 2011; De Veylder et al, 2011; Edgar et al, 2014). Although we cannot exclude long-term effects of cisplatin to promote endoreplication, we did not see a major increase in endoreplication levels in comparison with control plants when we analyzed cells of the root tips of 5-day-old wild-type plants grown for 24 h on media with 50 μM cisplatin. Likewise, a strong increase in endoreplication
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