Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants
2006; Springer Nature; Volume: 25; Issue: 23 Linguagem: Inglês
10.1038/sj.emboj.7601434
ISSN1460-2075
AutoresM. Endo, Yuichi Ishikawa, Keishi Osakabe, Shigeki Nakayama, Hidetaka Kaya, Takashi Araki, Kei‐ichi Shibahara, K. Abe, Hiroaki Ichikawa, Lisa Valentine, Barbara Höhn, Seiichi Toki,
Tópico(s)Plant tissue culture and regeneration
ResumoArticle16 November 2006free access Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants Masaki Endo Masaki Endo Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Graduate School of Life Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan Search for more papers by this author Yuichi Ishikawa Yuichi Ishikawa Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan Search for more papers by this author Keishi Osakabe Keishi Osakabe Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Shigeki Nakayama Shigeki Nakayama Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Hidetaka Kaya Hidetaka Kaya Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Takashi Araki Takashi Araki Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kei-ichi Shibahara Kei-ichi Shibahara Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Kiyomi Abe Kiyomi Abe Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Hiroaki Ichikawa Hiroaki Ichikawa Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Lisa Valentine Lisa Valentine Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Barbara Hohn Barbara Hohn Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Seiichi Toki Corresponding Author Seiichi Toki Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Masaki Endo Masaki Endo Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Graduate School of Life Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan Search for more papers by this author Yuichi Ishikawa Yuichi Ishikawa Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan Search for more papers by this author Keishi Osakabe Keishi Osakabe Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Shigeki Nakayama Shigeki Nakayama Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Hidetaka Kaya Hidetaka Kaya Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Takashi Araki Takashi Araki Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kei-ichi Shibahara Kei-ichi Shibahara Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Kiyomi Abe Kiyomi Abe Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Hiroaki Ichikawa Hiroaki Ichikawa Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Lisa Valentine Lisa Valentine Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Barbara Hohn Barbara Hohn Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Seiichi Toki Corresponding Author Seiichi Toki Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan Search for more papers by this author Author Information Masaki Endo1,2,‡, Yuichi Ishikawa1,3,‡, Keishi Osakabe1, Shigeki Nakayama1, Hidetaka Kaya4, Takashi Araki4, Kei-ichi Shibahara5, Kiyomi Abe1, Hiroaki Ichikawa1, Lisa Valentine6, Barbara Hohn6 and Seiichi Toki 1 1Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki, Japan 2Graduate School of Life Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan 3Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan 4Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan 5Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan 6Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. Tel.: +81 29 838 8450; Fax: +81 29 838 8450; E-mail: [email protected] The EMBO Journal (2006)25:5579-5590https://doi.org/10.1038/sj.emboj.7601434 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta ∼40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR—all factors that could also contribute to upregulation of HR frequency in fas mutants. Introduction In eukaryotic cells, the genomic DNA is highly compacted into chromatin through assembly with histone and nonhistone proteins. In proliferating cells, the bulk of the chromatin is assembled during DNA replication in the S phase of the cell cycle (Krude and Keller, 2001; Tyler, 2002; Verreault, 2003). Replication-specific nucleosome assembly is mediated by histone chaperones such as chromatin assembly factor 1 (CAF-1). CAF-1 was originally purified from nuclear extracts of human cells as a factor that supports the assembly of nucleosomes specifically onto replicating DNA in vitro (Smith and Stillman, 1989). CAF-1 mediates the first step of nucleosome assembly, that is, the deposition of H3/H4 histones onto replicating DNA (Smith and Stillman, 1989, 1991; Shibahara and Stillman, 1999; Tagami et al, 2004). CAF-1 is also involved in nucleosome assembly after nucleotide excision repair (NER; Ridgeway and Almouzni, 2000). CAF-1 is evolutionarily conserved, and homologs have been described in yeast, insects, plants, and vertebrates. In yeast, CAC1, CAC2, and CAC3 are counterparts of the p150, p60, and p48 subunits of human CAF-1. Despite the important role played by CAF-1 in nucleosome assembly during DNA synthesis, yeast cac mutants did not yield a lethal phenotype. However, increased UV sensitivity (Kaufman et al, 1997; Game and Kaufman, 1999), impaired gene silencing at telomeres (Monson et al, 1997; Enomoto and Berman, 1998) and at mating loci (Enomoto et al, 1997), and gross chromosomal rearrangements (Myung et al, 2003) were reported in such mutants. In higher eukaryotes, on the other hand, evidence for a more essential in vivo function of CAF-1 is accumulating. Ectopic expression of a dominant-negative form of the p150 subunit of CAF-1 caused severe early developmental defects in Xenopus laevis (Quivy et al, 2001), and induced S-phase arrest, accompanied by DNA damage and S-phase checkpoint activation, in human cells (Ye et al, 2003). Knockdown of the p60 subunit of CAF-1 by RNAi in human cells led to induction of cell death in proliferating, but not in quiescent, cells (Nabatiyan and Krude, 2004). In Arabidopsis thaliana, the CAF-1 subunits corresponding to the human subunits p150, p60, and p48 are encoded by FAS1, FAS2, and, most likely, MSI1, respectively (Kaya et al, 2001; Henning et al, 2003). fas1 and fas2 mutants of Arabidopsis were originally described as mutations causing stem fasciation, and abnormal phyllotaxy, leaf shape, root growth, and flower organ number (Reinholz, 1966; Leyser and Furner, 1992). These mutants display severely disturbed cellular and functional organization of both shoot apical meristem (SAM) and root apical meristem (RAM). They also show a varied pattern of distorted expression of both WUSCHEL and SCARECROW, which play key roles in the organization of SAM and RAM, respectively (Kaya et al, 2001). Thus, CAF-1 appears to be important for the maintenance of plant developmental gene expression patterns. Other than these alterations in postembryonic development, these mutants are viable. We speculated that delayed assembly of histones, expected as a consequence of a lack of CAF-1 activity, might lead to enhanced genomic instability in fas mutants. In this study, we used two different assays to measure DNA instability in a chromatin context: somatic homologous recombination (HR) and integration of the transferred DNA (T-DNA) of Agrobacterium tumefaciens. HR is a process that is used for precise DNA repair in somatic plant cells, whereas in meiosis it is used to generate novel distribution of genetic material between maternal and paternal chromosomes (Schuermann et al, 2005). T-DNA is a widely used tool for genetic engineering and plant insertional mutagenesis (Galbiati et al, 2000). Although we currently have very limited knowledge of the roles played by plant genes and proteins during this process, T-DNA integration is considered to use a nonhomologous end-joining (NHEJ)-related mechanism (Zupan et al, 2000; van Attikum et al, 2001; Friesner and Britt, 2003). Analysis of flanking sequence tags (FSTs) revealed that integration events are progressively less frequently observed towards the centromere (Brunaud et al, 2002), and also that about 40% of integration events occur in genes. These data suggested that chromatin structure can prevent T-DNA integration. In this study, we detected an ∼40-fold increase in the frequency of HR, as well as increased T-DNA integration, in fas mutants. To aid further understanding of these findings, we analyzed the transcription of DNA repair genes, the generation of DNA double-strand breaks (DSBs), and cell cycle progression in fas mutants. The results presented here suggested that delayed chromatin assembly could lead to prolonged exposure of not yet chromatinized DNA to enzymes capable of repairing DNA by either HR or NHEJ in plants. In addition, induced DNA DSBs and enhanced transcription of genes involved in HR at S phase could stimulate HR. Results The frequency of HR is strongly elevated in fas mutants We used an HR repair assay that allows recombination events to be visualized and scored by histochemical staining for a reconstituted recombination substrate locus (Swoboda et al, 1994; Schuermann et al, 2005). The constructs used as substrates for HR contain parts of the β-glucuronidase (GUS) gene in direct or inverted orientation (Figure 1A), which can recombine to reconstitute a functional GUS gene. fas1-2 (ecotype Nossen) and fas2-1 (ecotype Landsberg erecta (Ler)) plants were crossed to Arabidopsis lines (ecotype Columbia (Col)) carrying such recombination substrates, and F3 progeny plants were monitored for lines homozygous for the recombination reporter construct as well as for the fas1 and fas2 mutation. Mutants in either fas1 or fas2 resulted in around 40-fold more GUS recombination spots than in wild-type plants (Figure 1B–E), independent of the relative orientation of the truncated recombination target sequences, and regardless of which CAF-1 subunit was mutated. To confirm that the difference in HR frequency was not due simply to the heterogeneous genetic background of the parent plants, we analyzed HR frequency of FAS2 RNAi knockdown plants as well as a T-DNA tagging mutant of FAS2 (fas2-4), both of which are in a Col background. Both these plant lines also showed enhanced HR frequency compared to wild-type Columbia (Supplementary Figure S1). Figure 1.Genomic flexibility, as measured by intrachromosomal HR and T-DNA integration, is increased in fas mutants. (A) Recombination marker constructs. The β-glucuronidase (GUS; uidA) sequences have an overlap (indicated by ‘U’) either in the direct (left) or inverted (right) orientation. Recombination (indicated by ‘x’) between the two overlapping sequences produces a functional GUS gene. (B, C) Visualization of recombination events by histochemical GUS staining of leaves from a FAS1 control (B), and a fas1-2 plant homozygous for the inverted repeat-type recombination reporter (GU-US/GU-US) (C). An arrowhead in (B) indicates GUS-positive cells. (D, E) Frequency distribution histograms showing the proportions of plants with a given number of blue GUS spots in the direct repeat (D) and inverted repeat (E) populations. (F, G) Mutants fas1-2 and fas2-2 (ecotype Nossen), fas2-1 (ecotype Ler), and fas2-4 (ecotype Col), and the corresponding wild-type plants were inoculated with A. tumefaciens. (F) Plates showing growth of roots and tumors of Nossen, and its mutants fas1-2 and fas2-2, photographed 1 month after infection. (G) Efficiency of T-DNA integration as represented by the percentage of root segments that produced tumors. Error bars indicate standard error (s.e.). Data (means±s.e.) were taken from 10 plants of each type. Download figure Download PowerPoint T-DNA integration is enhanced in fas mutants The mechanism of in vivo integration of T-DNA into plant DNA represents a special case of a NHEJ process. NHEJ is the main pathway used by higher eukaryotic organisms to repair DSBs in DNA. This repair mechanism is usually accomplished with concomitant changes at the junction sequence, and is thus error prone (Lees-Miller and Meek, 2003). The efficiency of T-DNA integration can be assessed using a root tumorigenesis assay (Nam et al, 1999). Root transformation with Agrobacterium A208 results in large green tumors on the roots. Indeed, increased numbers of tumors were observed on roots of fas1 and fas2 mutants infected with Agrobacterium compared to roots of the respective wild-type ecotypes (Figure 1F and G). When we analyzed transient expression of GFP following Agrobacterium-mediated root transformation, no difference in GFP expression between wild-type and fas mutants was observed (data not shown), suggesting that CAF-1 depletion does not increase T-DNA transmission from Agrobacterium to plant nuclei. It is interesting to note that ecotypes naturally more refractory to T-DNA integration, such as Ler and, especially, Nossen, reacted more strongly to the mutation than Col, which is already very sensitive to T-DNA integration in the wild-type context. Enhanced transcription of genes involved in HR in fas mutants The results of the two experiments described above could be linked to defects in nucleosome assembly according to various, not necessarily mutually exclusive, scenarios: (i) the expression of repair enzymes is upregulated, (ii) there is an increased level of breaks in the DNA that can be repaired by HR and NHEJ activities, and (iii) repair enzymes, due to a lack, or delayed assembly, of nucleosomes, could have easier access to breaks in DNA in need of repair. To test the hypothesis that an increased level of transcription of repair enzymes might contribute to enhanced rates of HR and T-DNA integration, we measured the transcription levels of several such genes by real-time PCR. As shown in Figure 2A, AtRAD51, which plays a central role in HR repair (Doutriaux et al, 1998), was found to be upregulated 4–5-fold in fas mutants. Transcription of AtRAD54 (a homolog of the yeast RAD54 gene, K Osakabe et al, 2006) was slightly induced in the mutants, whereas transcription of genes commonly grouped in the NHEJ pathway (AtKU70, AtKU80, and AtLIG IV) was unchanged, as was transcription of AtLIG I, which encodes a DNA replication factor. The results of Western blot analysis also indicated that the amount of AtKu70 protein remained constant in fas mutants (Supplementary Figure S2). Figure 2.Elevated transcription of HR repair genes, and increased DNA damage in fas mutants. (A) Transcript levels of the repair genes indicated in fas1-2 and fas2-2 mutants relative to wild-type Nossen, as determined by real-time quantitative PCR. Error bars indicate standard error (s.e.). (B) Transcript levels of the repair genes indicated at 6 and 12 h after γ-irradiation in wild-type Nossen as determined by microarray analysis. (C–E) Comet images of intact and fragmented nuclear DNA from wild-type Nossen. (C) An almost intact nucleus (short tail). (D) A damaged nucleus (long tail). (E) A severely damaged nucleus (long fragmented tail). Original images are shown as white colored comets in the right corner panels of (C) and (D), and in the lower panel of (E). DNA intensity is indicated by gradation of color. (F) Schematic representation of a comet. (G) Statistical analysis of a comet assay. The level of DNA DSBs in the nucleus is represented as the tail moment, defined as the product of comet tail length and the fraction of total DNA in the tail (see Materials and methods). Error bars indicate the s.e. values of analyzed cells. Download figure Download PowerPoint Interestingly, the same genes that were transcriptionally upregulated in the fas mutants (AtRAD51 and AtRAD54; Figure 2A) were also found to have a higher steady-state level of transcription in wild-type Arabidopsis plants exposed to γ-irradiation (Figure 2B). Increased level of DNA DSBs in fas mutants The experiments described above suggested the increased presence of DNA DSBs in fas mutants. To directly monitor the extent of DSBs in the DNA of wild-type and fas mutants, we attempted to quantify DNA DSBs by comet assay (Menke et al, 2001), which can indicate DNA damage in individual cells. A schematic representation of a comet assay is shown in Figure 2F. The amount of DNA in the comet tail separated from intact nuclear DNA in an electric field correlates with the number of breaks in the nuclear DNA (Menke et al, 2001). Figures 2C–E show typical comet assays of wild-type Arabidopsis nuclei with different amounts of DNA damage. As shown in Figure 2G, a small but significant increase in the number of DSBs was observed in fas mutants compared to wild-type plants. Severely damaged and fragmented nuclei (Figure 2E) were found more frequently in fas mutants (12.8 and 9.1% of the total number of cells counted in fas1-2 and fas2-2) than in wild-type (1.7% of total cells counted) but such nuclei were not included in the statistical analysis. Therefore, the relative level of DNA DSBs in fas mutants should be greater than that shown in Figure 2G. One of the earliest known responses to DSB induction is the phosphorylation of thousands of molecules of the histone variant H2AX at the site of the break (Rogakou et al, 1998). In Arabidopsis, induction of phosphorylated H2AX (known as γ-H2AX) in an irradiation-dose-dependent manner, and its subsequent disappearance through DNA DSB repair have been demonstrated (Friesner et al, 2005). Quantification of γ-H2AX in wild-type and fas mutants by Western blot analysis revealed a small induction of γ-H2AX in fas mutants compared to wild-type (1.3-fold in fas1-2 and 2.1-fold in fas2-2) (Supplementary Figure S3), thus supporting the comet assay data. fas mutants show increased sensitivity to DNA-damaging treatments fas mutants were expected to be more sensitive to DNA-damaging stresses compared to wild-type plants due to the intrinsically higher level of DNA DSBs. Thus, we next investigated the γ-ray sensitivity of fas1 and fas2 mutants. As shown in Figures 3A–C, root growth of Arabidopsis seedlings was inhibited by γ-irradiation in a dose-dependent manner. This inhibition was greater in fas mutants than in wild-type plants, regardless of ecotype. We also assessed the development of true leaves in γ-irradiated plants (Figure 3D). fas1 and fas2 mutants exhibited increased yellowing of the cotyledons, resulting in death, following 600 Gy irradiation. In contrast, true leaves from wild-type plants did emerge. In fas1-2, 60, 20, 20, 10, and 0% of plants produced true leaves after 200, 300, 400, 500, and 600 Gy of γ-irradiation. In fas2-2, 70, 60, 50, 30, and 20% of plants produced true leaves after 200, 300, 400, 500, and 600 Gy of γ-irradiation. Under our experimental conditions, all plants could produce true leaves after 200–600 Gy of γ-irradiation in wild-type Nossen. Similarly, fas1 and fas2 mutants are more sensitive to UV-C irradiation than wild-type plants (see Supplementary Figure S4). Figure 3.fas mutants show increased sensitivity to DNA-damaging treatments. (A, B) Phenotype of seedlings following exposure to the doses of γ-irradiation is indicated. (A) Wild-type Nossen (top), Nossen background mutants fas1-2 (middle) and fas2-2 (bottom). (B) Wild-type Ler (top) and Ler background mutant fas2-1 (bottom). (C) Relative root growth of fas1-2, fas2-2, and fas2-1 mutants and wild-type plants after exposure to the doses of γ-irradiation indicated. The average root length of nonirradiated plants in each case was taken as 100%. Error bars indicate s.e. Data are means±s.e., and represent the results of three independent experiments. (D) Development of true leaves of fas1-2, fas2-2, and fas2-1 mutants and the corresponding wild-type plants after γ-irradiation (600 Gy). Download figure Download PowerPoint Cell cycle regulation in fas mutants An essential step in the completion of S phase of the cell cycle is the reassembly of histone onto newly replicated DNA. CAF-1 is involved in this process. To analyze the effect of CAF-1 depletion on cell cycle progression, we analyzed the proportion of cells in each phase of the cell cycle by flow cytometry. Nuclei from the true leaves of 9-day-old seedlings showed an increase in 4C and a decrease in 2C cells in fas1-2 and fas2-2 mutants (Figure 4A and B). These results suggest an increased frequency of nuclei in G2/M phase in fas mutants. Figure 4.Aberrant cell cycle regulation in fas mutants. (A) Flow cytometric analysis of true leaves in 9-day-old Nossen, and Nossen background fas1-2 and fas2-2 mutants. (B) Calculated proportion of multiploid cells in 9-day-old Nossen, and Nossen background fas1-2 and fas2-2 mutants. (C) Transcription of cyclin genes in fas1-2 and fas2-2 mutants as determined by microarray analysis. (D) Transcript levels of cyclin genes 6 and 12 h after γ-irradiation in wild-type Nossen as determined by microarray analysis. Download figure Download PowerPoint To further investigate cell cycle progression in fas mutants, we analyzed transcription of cyclin genes using microarrays (Figure 4C). In this assay, transcription of the mitotic cyclin AtCYCB1;1 (At4g37490, B-type cyclin gene) was drastically increased in fas mutants. The Arabidopsis mitotic cyclin CYCB1;1 product is reported to accumulate only around the time of the G2/M transition (Doerner et al, 1996; Shaul et al, 1996). Interestingly, expression of this cyclin was also strongly induced by γ-irradiation (Figure 4D), suggesting the crucial role of a G2 retardation for DNA DSB repair following γ-irradiation. To confirm the results of microarray analysis and to further investigate the effects of CAF-1 depletion on cell cycle regulation, especially tissue-specific effects, we examined the expression of AtCYCB1;1∷GUS. Mitotic cyclin turnover requires a short peptide motif known as the ‘destruction box’ (King et al, 1996). Transcriptional and post-translational regulation together restricts the accumulation of mitotic cyclins to G2 and M phase of the cell cycle. Arabidopsis Col plants transformed with AtCYCB1;1∷GUS (Colon-Carmona et al, 1999) were crossed with fas mutants. Figure 5 shows an example of AtCYCB1;1∷GUS expression in fas2-4 and wild-type Col. As shown in Figures 5A–C, cells expressing GUS activity appeared sporadically only in the root tip of wild-type Col. In contrast, significantly large numbers of cells in the stems, flower buds, leaves, and root tips of the fas2-4 mutant showed strong GUS activity (Figure 5D–F). Enhanced expression of AtCYCB1;1∷GUS was also detected regardless of ecotype and of which CAF-1 subunit was mutated (Supplementary Figure S5). Figure 5.Histochemical assay of the AtCYCB1;1∷GUS reporter. GUS staining patterns of Col (A–C) and Col background fas2-4 mutant (D–F) are shown. (A, D) Close-up of flower buds. (B, E) GUS staining of true leaves. Arrowheads in (E) indicate GUS-positive cells in a fas2-4 leaf. (C, F) Close-up of root tips. Download figure Download PowerPoint Discussion Hyper-recombination of genomic DNA in fas mutants In the present study, mutants in either fas1 or fas2 exhibited around 40-fold more recombination events than observed in wild-type plants (Figure 1A–E). We interpret this to mean that it is the complete CAF-1 complex, and not any one individual subunit, that maintains HR at a low level in wild-type plants. In this context, stimulated intrachromosomal recombination was reported in a yeast cac1 mutant (Prado et al, 2004), but in this case the increase was only 2–3-fold. These differences could be connected to the fact that HR efficiency in yeast is already high under normal conditions. In vertebrate systems, histone H3 comes in two major forms, histone H3.1 and histone H3.3. The former is loaded onto replicated DNA via CAF-1, whereas H3.3 replaces H3.1 in a postreplicative pathway via HIRA (Tagami et al, 2004). Interestingly, yeast has only the H3.3 version of histone H3 (Ahmad and Henikoff, 2002), hence CAF-1 might not be essential in yeast. The relative importance of CAF-1 between plants and yeast could thus result in a difference in the ratio of HR enhancement. It will be of interest to investigate HR in vertebrates with a similar assay. CAF-1 was reported to be involved in the maintenance of epigenetic state in yeast (Monson et al, 1997; Enomoto and Berman, 1998). Therefore, the increased number of GUS spots observed in fas mutants could be explained by the release of transcriptional gene silencing (TGS) of the GUS gene. However, based on the following arguments, we judge the increased number of GUS spots observed in fas mutants to be due mainly to hyper recombination of the recombination substrate: we tested two different loci for indication of the hyper-recombination phenotype and both showed the same behavior. A hygromycin-resistance gene (hpt) was located between the two disrupted (but partially overlapping) GUS gene fragments (see Figure 1A) and the plants used in this assay showed stable hygromycin resistance. Hence, silencing of the GUS locus is unlikely. Furthermore, recently it has been reported that TGS of a silent GUS transgene was partially, but not totally de-repressed in fas mutants (Ono et al, 2006). These data thus concur with our prediction that fas mutants indeed exhibit an increased level of genome instability, as measured by intrachromosomal HR. A mutation in the yeast linker histone HHO1 led to an increased frequency of HR (Downs et al, 2003); partial depletion of histone H4 gave a similar phenotype (Prado and Aguilera, 2005). As CAF-1 assembles histones H3 and H4 following DNA replication (Smith and Stillman, 1989; Shibahara and Stillman, 1999; Tagami et al, 2004), depletion of CAF-1 and partial loss of histone H4 could enhance HR by similar mechanisms. Increased accessibility of the T-DNA/protein complex to genomic DNA in fas mutants Using a root tumorigenesis assay, we detected increased T-DNA integration in fas mutants (Figure 1F and G). As T-DNA integration occurs mainly through non-HR mechanisms, it is most likely that NHEJ proteins are required for the process of T-DNA integration. In fact, ku80-mutant Arabidopsis plants are defective in T-DNA integration in somatic cells, whereas Ku80-overexpressing plants exhibit increased susceptibility to Agrobacterium infection (Li et al, 2005). However, real-time PCR analysis failed to detect increased transcription of NHEJ pathway genes (AtKU70, AtKU80, and AtLIG IV) in fas mutants (Figure 2A). Furthermore, the protein level of Ku70 was unchanged in wild-type and fas mutants (Supplementary Figure S2). Therefore, the increased levels of T-DNA integration observed in fas mutants might reflect an increased level of genome instability rather than enhanced expression of NHEJ pathway genes. The root tumorigenesis
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