A homologue of the breast cancer-associated gene BARD1 is involved in DNA repair in plants
2006; Springer Nature; Volume: 25; Issue: 18 Linguagem: Inglês
10.1038/sj.emboj.7601313
ISSN1460-2075
AutoresWim Reidt, Rebecca Wurz, Kristina Wanieck, Chu Hoàng Hà, Holger Puchta,
Tópico(s)BRCA gene mutations in cancer
ResumoArticle7 September 2006free access A homologue of the breast cancer-associated gene BARD1 is involved in DNA repair in plants Wim Reidt Wim Reidt Search for more papers by this author Rebecca Wurz Rebecca Wurz Search for more papers by this author Kristina Wanieck Kristina Wanieck Search for more papers by this author Hoang Ha Chu Hoang Ha Chu Search for more papers by this author Holger Puchta Corresponding Author Holger Puchta Botanisches Institut II, Universität Karlsruhe, Karlsruhe, Germany Search for more papers by this author Wim Reidt Wim Reidt Search for more papers by this author Rebecca Wurz Rebecca Wurz Search for more papers by this author Kristina Wanieck Kristina Wanieck Search for more papers by this author Hoang Ha Chu Hoang Ha Chu Search for more papers by this author Holger Puchta Corresponding Author Holger Puchta Botanisches Institut II, Universität Karlsruhe, Karlsruhe, Germany Search for more papers by this author Author Information Wim Reidt, Rebecca Wurz, Kristina Wanieck, Hoang Ha Chu and Holger Puchta 1 1Botanisches Institut II, Universität Karlsruhe, Karlsruhe, Germany *Corresponding author. Botanisches Institut II, Universität Karlsruhe, Kaiserstrasse 12, 76128 Karlsruhe, Germany. Tel.: +49 721 608 3833; Fax: +49 721 608 4874; E-mail: [email protected] The EMBO Journal (2006)25:4326-4337https://doi.org/10.1038/sj.emboj.7601313 Correction(s) for this article A homologue of the breast cancer-associated gene BARD1 is involved in DNA repair in plants18 April 2007 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info hBRCA1 and hBARD1 are tumor suppressor proteins that are involved as heterodimer via ubiquitinylation in many cellular processes, such as DNA repair. Loss of BRCA1 or BARD1 results in early embryonic lethality and chromosomal instability. The Arabidopsis genome carries a BRCA1 homologue, and we were able to identify a BARD1 homologue. AtBRCA1 and the putative AtBARD1 protein are able to interact with each other as indicated by in vitro and in planta experiments. We have identified T-DNA insertion mutants for both genes, which show no visible phenotype under standard growth conditions and are fully fertile. Thus, in contrast to animals, both genes have no indispensable role during development and meiosis in plants. The two single as well as the double mutant are to a similar extent sensitive to mitomycin C, indicating an epistatic interaction in DNA crosslink repair. We could further demonstrate that in Arabidopsis BARD1 plays a prominent role in the regulation of homologous DNA repair in somatic cells. Introduction Germline mutations of the hBRCA1 (breast cancer susceptibility 1) gene are known to be responsible for about 50% of all inherited breast cancer cases (Miki et al, 1994). The human BRCA1 gene codes for an 1863 amino acids (aa) long nuclear protein with two functionally important motifs. The first motif is located at the N-terminus of the protein and codes for a RING-finger domain, consisting of 40–60 aa. Many RING finger containing proteins function as ubiquitin E3 ligase (Wu et al, 1996; Joazeiro and Weissman, 2000). The second motif is located at the C-terminus and encodes two repeats of approximately 80 aa. These repeats were designated as Breast cancer C-terminal repeats (BRCT; Callebaut and Mornon, 1997; Koonin et al, 1996). These BRCT domains are present in a large number of cell cycle checkpoint proteins ranging from bacteria to humans (Koonin et al, 1996; Callebaut and Mornon, 1997). Both RING and BRCT domains of hBRCA1 are well conserved and serve as common sites for missense mutations that predispose women to early-onset breast cancer (Ruffner et al, 2001; Rodriguez et al, 2004). Protein interaction studies using either the RING or BRCT domain of BRCA1 identified several interacting proteins (Jensen et al, 1998; Yarden and Brody, 2001). Interestingly, a protein found to interact with the N-terminal RING domain contained itself both a RING as well as two BRCT domains similar to BRCA1 (Wu et al, 1996). As further studies demonstrated that both proteins are able to form a heterodimer through their common N-terminal RING domain, this protein was designated BARD1, breast cancerassociatedRINGdomain (Meza et al, 1999; Joukov et al, 2001). This hBRCA1/hBARD1 heterodimer complex functions as an E3 ubiquitin ligase that catalyses the synthesis of polyubiquitin chains (reviewed by Baer and Ludwig, 2002). DNA damage poses a continuous threat to genomic integrity in eukaryotic cells. A particularly lethal form of DNA damage is the DNA double-strand break (DSB). Cells have two major pathways for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ) (reviewed by Puchta, 2005). Although NHEJ is a process in which the ends of a DSB might be modified, HR precisely restores the continuity of a broken DNA molecule using an intact and homologous DNA strand as template. For a decade, multiple analyses have been performed to elucidate the biological role of BRCA1. Evidence for the involvement of BRCA1 in the repair of DSB originates from its association with hRAD51 (Scully et al, 1997a), and from the formation of foci at sites of DSBs after genotoxic stress (Scully et al, 1997b; Paull et al, 2000). Disruption of BRCA1 in mice results in embryonic lethality that is accompanied by growth retardation, apoptosis, cell cycle defects and genetic instability (Gowen et al, 2000). Taken together, these results demonstrate a very important role for BRCA1 in promoting HR and thus in maintaining genomic integrity. In contrast to BRCA1 very few and partially indirect functional studies on BARD1 homologues were performed. Besides its function as E3 ubiquitin ligase in a complex with BRCA1, some studies indicated that the protein might also be involved in homologous DSB repair (Westermark et al, 2003; Stark et al, 2004). Recently, studies on a BARD1 homologue in Caenorhabditis elegans showed that depletion of the BARD1 protein resulted in germination defects and radiation sensitivity (Boulton et al, 2004). Until 2003 orthologues of BRCA1 were only identified in other animal genomes, for example, C. elegans and Xenopus laevis (Joukov et al, 2001; Boulton et al, 2004). Surprisingly, Lafarge and Montane identified in 2003 a BRCA1 orthologue in the genome of the model plant Arabidopsis. Similar to its orthologue from humans, this protein has the characteristic RING and BRCT domains. Furthermore, it was shown that the transcription of the AtBRCA1 gene was strongly induced by γ-irradiation (Lafarge and Montane, 2003). However, the study did not address the biological function of the protein in plants. We have now been able to identify a hBARD1 homologue in Arabidopsis and in the following we characterise the biological role of AtBARD1 and AtBRCA1 in plants. Results Identification of a hBARD1 homologue in A. thaliana The characteristic feature of both hBRCA1 and hBARD1 is the presence of a conserved RING as well as two BRCT domains. Orthologues of hBRCA1 and hBARD1 in Mus musculus, C. elegans or X. laevis display a similar domain structure (Szabo et al, 1996; Joukov et al, 2001; Boulton et al, 2004). To identify putative BARD1 homologues in the Arabidopsis genome, a database search was carried out in TAIR-BLASTP using hBARD1 as template (NP000456). This search resulted in two significant hits: At4g21070, which had previously been classified as the hBRCA1 homologue of Arabidopsis (Lafarge and Montane, 2003), and At1g04020. The homology of AtBRCA1 to the hBARD1 protein is restricted to the previously mentioned conserved RING and BRCT domains. However, At1g04020 has additional homology to hBARD1 outside the RING and BRCT domains, in total 22% amino-acid identity and 38% similarity (Figure 1A and B). We therefore assumed that At1g04020 might be the BARD1 homologue of Arabidopsis. Figure 1.Gene structure of AtBARD1 and comparison of the AtBARD1 and hBARD1 proteins. (A) A schematic representation of the AtBARD1 intron–exon structure. Exons are represented by grey boxes, introns by black bars. In total, the AtBARD1 gene counts 13 exons, the gene has a length of 3436 bp encoding a protein of 714 aa. The AtBARD1 protein has a similar structure as the hBARD1 protein, also containing a conserved RING domain (black regions) and two BRCT domains (light grey regions). Both proteins have an identity of 22% and a similarity of 38%. (B) Protein sequence alignment of AtBARD1 against hBARD1. Identical amino acids are shaded black whereas similar amino acids are shown in grey. Conserved RING and BRCT domain structures are indicated by black and light grey frames, respectively. Download figure Download PowerPoint Using mRNA from Arabidopsis flowers as template, the cDNA from At1g04020 could be amplified by polymerase chain reaction (PCR). By RACE-PCR with nested gene-specific primers, 5′ and 3′ ends were obtained. The ORF of full-length AtBARD1 has a total length of 2145 bp, contains 13 exons, and codes for a protein of 714 aa (Figure 1A). A cDNA clone (BX815982) from the GenBank confirmed this structure. The ORF of AtBRCA1 was also determined by RACE-PCR. In line with cDNA clones from SALK (U24692, R24692 and AF515728), we identified the ORF of full-length AtBRCA1 consisting of 2826 bp, containing 14 exons and coding for a protein of 941 aa. This is in contrast to the original report of Lafarge and Montane (2003) who identified the ORF of full-length AtBRCA1 consisting of 4485 bp and 15 exons. The first exon postulated by Lafarge and Montane (2003) is part of another gene rather than the ORF of AtBRCA1. In plants it was demonstrated before that some genes coding for proteins involved in nucleotide metabolism and DNA repair can be induced by DNA damage, among them AtBRCA1 (e.g. Chen et al, 2003; Lafarge and Montane, 2003). To characterise a possible correlation between the expression of AtBRCA1 and AtBARD1 2-week-old seedlings were irradiated by γ-ray (100 Gy) and the transcript amount of both genes was measured after 1 h by quantitative real-time PCR (Figure 2A). As reported previously, a strong induction of the AtBRCA1 transcript could be detected. In contrast, no significant change of the mRNA level of AtBARD1 was found. Additionally, the expression of both genes in different tissues of 6–8-week-old Arabidopsis plants was analysed. RNA from roots, rosette leaves, inflorescence, young cauline leaves, flowers and siliques was isolated and the transcript amount of both genes was measured via real-time PCR. Higher amounts of mRNA of both AtBRCA1 and AtBARD1 could be detected in flowers and siliques. The expression in roots, rosette leaves, inflorescence and young cauline leaves was low (Figure 2B). Thus, in contrast to the application of genotoxic stress, the expression pattern of both genes in different organs correlated well, hinting to a functional interaction. Figure 2.Expression analysis of AtBARD1 and AtBRCA1 in Arabidopsis. (A) The expression of AtBARD1 and AtBRCA1 was analysed by relative quantification using real-time PCR 1 h after irradiation by γ-ray. Transcription level ratio of AtBARD1 and AtBRCA1 is given in relation to actin mRNA and the mRNA of the respective untreated seedlings, and is the mean of six different reactions ±s.d. White bars, AtBARD1; grey bars, AtBRCA1. (B) The expression pattern of AtBARD1 and AtBRCA1 in different plant tissues was analysed by relative quantification using real-time PCR. RNA from roots, rosette leaves, inflorescence, cauline leaves, flowers and siliques of soil-grown plants was analysed. Expression of AtBARD1/AtBRCA1 is given relative to actin mRNA levels and is the mean of six different reactions±s.d. Similar results were obtained in independent experiments. Download figure Download PowerPoint Protein–protein interaction between AtBARD1 and AtBRCA1 To test whether AtBRCA1 and the putative AtBARD1 protein are also able to interact directly, a two-hybrid analysis was performed. First, it was tested whether AtBRCA1 and AtBARD1 contained an activation domain. It was previously demonstrated that this is the case for the hBRCA1 protein, whereas so far this has not been reported for the hBARD1 protein (Welcsh et al 2002). With the help of the LexA-based yeast two-hybrid system, we could clearly demonstrate that the full-length AtBARD1 protein contained an autoactivation domain (Figure 3A). Unfortunately, no consistent results were obtained using the full-length AtBRCA1 protein. This might reflect the presence of a weak transcriptional activation domain. Thus, in this assay it was only possible to use truncated versions of AtBRCA1 or AtBARD1 as bait. An N-terminal fragment of AtBRCA1 coding for the first 59 aa and containing the RING domain did not display any autoactivation and was used as bait. As prey the full-length AtBARD1 protein and a C-terminal AtBARD1 fragment containing the BRCT repeats but missing the RING domain were constructed. Indeed, an interaction of the RING domain of AtBRCA1 with the full-length AtBARD1 protein could be demonstrated (Figure 3A), whereas no interaction of the RING domain of AtBRCA1 with the C-terminal part of the AtBARD1 protein was found. Unfortunately, we failed to detect an interaction using the RING domain of AtBARD1 as bait and the complete AtBRCA1 protein (result not shown). However, it is not uncommon in two-hybrid analysis that only certain bait and prey combinations result in detectable interactions (Uetz et al, 2000). Figure 3.Characterisation of the AtBARD1 and AtBRCA1 interaction by yeast two-hybrid assay and by BiFC in transiently transfected mustard seedlings. (A) Yeast two-hybrid assay. Different constructs of AtBRCA1 or AtBARD1 were used either as bait (DNA-BD) or prey (DNA-AD) and tested for their ability to activate the lacZ reporter gene (X-gal) and the nutritional marker gene leucine (Leu). The full-length protein AtBARD1 was, when fused to a DNA-binding domain (DNA-BD), able to activate the lacZ reporter gene as well as the leucine reporter gene. The use of AtBRCA1 as bait led to inconsistent results. A truncated version of AtBRCA1, containing the first 59 N-terminal aa (representing the RING domain) interacted with the complete AtBARD1 protein fused to the activation domain and resulted in the activation of the lacZ reporter gene and the leucine reporter gene. No interaction could be demonstrated between the AtBRCA1 RING domain and a C-terminal part of AtBARD1 (AtBARD1 C-T). Furthermore, none of the single used constructs was able to activate transcription. Blue staining of the yeast colonies appeared within 30 min for the AtBARD1 protein, to up to 2 h for the AtBRCA1 AtBARD1 interaction, whereas the growth of yeast colonies on LEU lacking medium was determined after 2 days. (B–I) (B′–I′) and (B″–I″) BiFC analysis in transiently transfected mustard seedlings. The pictures B–I show an YFP signal in case of a protein interaction, in the nucleus of a representative cell, owing to the restoration of the YFP complex. The pictures B′–I′ show a CFP signal from a cotransfected nuclear marker. The pictures B″–I″ show the same cells as in (B–I) and (B′–I′), respectively, by bright field microscopy. Bars=20 μm. B, B′ and B″ AtBRCA1 (YC) and AtBARD1 (YN); C, C′ and C″ AtBRCA1 (YC) and empty vector pMAV-GW-YN; D, D′ and D″ AtBRCA1 RING (YC) and AtBARD1 (YN); E, E′ and E″ AtBRCA1 RING (YC) and empty vector pMAV-GW-YN; F, F′ and F″ AtBRCA1 C-terminus (YC) and AtBARD1 (YN); G, G′ and G″ AtBARD1 (YN) and empty vector pMAV-GW-YC; H, H′ and H″ ASK1 (YN) and EID1 (YC), positive control; I, I′ and I″ ASK1 (YN) and EID1ΔF (YC), negative control. Download figure Download PowerPoint Interaction of AtBARD1 and AtBRCA1 in planta To further sustain our observation that AtBRCA1 and AtBARD1 interact, in vivo studies were carried out. We used a well-established method of bimolecular fluorescence complementation (BiFC; Hu et al, 2002) for the in vivo detection of protein–protein interactions, namely the split YFP system (Stolpe et al, 2005). Briefly, the assay is based on the observation that a N- (YN) and a C-terminal (YC) fragment of the yellow fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Two ORFs, driven by a double 35S promoter, are fused on separate plasmids to the respective YFP fragments; next, both constructs are brought into a plant cell for expression and the interaction of the fusion proteins can be monitored via epifluorescence microscopy. To confirm the possible interaction between AtBRCA1 and AtBARD1, the full-length AtBRCA1 ORF, the first N terminal 88 aa coding for the AtBRCA1 RING domain and the last 797 C-terminal aa of the ORF of AtBRCA1 were fused to the C-terminal part of the YFP protein. The full-length AtBARD1 ORF was fused to the N-terminal part of the YFP protein. Next, the different constructs, together with a plasmid containing the CPRF2 protein (common plant regulatory factor 2) fused to CFP as nuclear marker (e.g. Figure 3B′ and C′), were transiently expressed after particle bombardment in etiolated mustard seedlings. As positive control, the ASK1 protein fused to the N-terminus of YFP, and an EID1-YFP-C-terminal fusion was used (Figure 3H). EID1 and ASK1 are interacting proteins of the Skp1-Cullin-F-box-protein ubiquitin ligase that targets proteins for degradation and functions as a negative regulator in phytochrome A-specific light signalling. The negative control was a deleted version of the EID1 (EID1ΔF) protein not able to interact with ASK1 (Figure 3I; Stolpe et al, 2005). As further controls AtBRCA1 or AtBARD1 constructs fused with the N- or C-terminal part of the YFP protein, respectively, were used together with the respective pMAV-GW-YN and pMAV-GW-YC empty vectors. After an overnight incubation period, the seedlings were screened for the presence of an YFP signal. Routinely, 1–5 transfected cells per seedling were obtained. The results are based on at least two independent experiments using four mustard seedlings for each transfection. For each single combination, the results were uniform, that is, besides the CFP signal, either in all or in none of the transfected cells an YFP signal could be detected. Not only in case of the full-length ORF of AtBRCA1 combined with the complete AtBARD1 protein an YFP signal could be detected (Figure 3B), but also in the AtBRCA1 RING domain and the AtBARD1 protein (Figure 3D). No YFP signal was observed when the C-terminus of AtBRCA1 was coexpressed with the AtBARD1 protein (Figure 3F). No YFP signal could be obtained when combinations of the single constructs of AtBRCA1 and AtBARD1 with the pMAV-GW-YN and pMAV-GW-YC empty vectors were used (Figure 3C, E and G). Taken together, our experiments clearly demonstrate that AtBARD1 and AtBRCA1 are able to interact, and that this interaction is mediated by the AtBRCA1 RING domain. This is in line with our experiments from the two-hybrid system. Mutant atbard1 plants are phenotypically normal but sensitive to mitomycin C Functional studies were necessary in order to elucidate the biological role of the BARD1 homologue in plants. The putative AtBARD1 gene sequence was used to screen the sequence database of T-DNA insertion mutants on the SIGnAL webpage (Salk Institute Genomic Analysis Laboratory; Alonso et al, 2003). Two atbard1 T-DNA mutant lines were identified. The respective plants were obtained, propagated, and homozygous individuals of the respective insertions could be identified. The insertion sites were determined in detail by PCR. Figure 4 provides a detailed characterisation of the T-DNA insertions of AtBARD1. Figure 4.Schematic structure of the AtBARD1 gene and its T-DNA insertions. (A) The AtBARD1 gene consists of 13 exons. Regions coding for the RING and BRCT domain are indicated in black and light grey, respectively. Two T-DNA insertions were identified. One insertion is located in the first intron, and denominated atbard1-1 whereas the second insertion is located in the third exon, and denominated atbard1-2. (B) An overview of the precise locations of the T-DNA inserts in the AtBARD1 gene. Intron sequences are displayed as lower-case letters, exon sequences as capital letters, and T-DNA border sequences are underlined (LB: left border). (C) Semiquantitative RT–PCR on different regions of the AtBARD1 gene. Primer pairs were used that bind in front of (a+b), across (c+d) and after (e+f) the T-DNA insertions. The β-tubulin gene was taken as control. WT: wild type. Download figure Download PowerPoint The two atbard1 T-DNA insertions are located at the beginning of the gene. Both insertions carry left T-DNA borders at their ends, indicating the integration of a double T-DNA insert in inverted orientation. The first insertion, SALK_097601, atbard1-1, is present in the first intron and results in a deletion of 18 nucleotides. The second insertion, SALK_031862, atbard1-2, is located in the third exon, which codes for the N-terminal RING domain and leads to a deletion of 5 nucleotides within the coding sequence (Figure 4B). In order to assess the expression level of AtBARD1 in the homozygous T-DNA lines, reverse transcription–polymerase chain reaction (RT–PCR) experiments were performed with homozygous mutants using primer pairs binding in front, across and after the insertions (Figure 4A and C). In case of both lines, expression of an mRNA before the insertion could be demonstrated (Figure 4C). With primers spanning across the insertions, we were not able to amplify any product for both alleles. An expression after the insertion was detected for atbard1-1. In contrast, no expression could be found for atbard1-2, indicating that this allele most probably represents a 'true' null atbard1 mutation. In comparison to wild-type plants, all plant lines homozygous for the respective insertions did not differ in their phenotypes when grown under standard conditions. However, when challenged with the DNA crosslinking agent mitomycin C (MMC), the T-DNA insertion mutants showed a more sensitive phenotype as compared to wild-type seedlings (Figure 5). The mutant seedlings were smaller and less viable. Interestingly, the line atbard1-2 showed a slightly stronger phenotype after treatment with MMC than atbard1-1. Other mutagenic treatments with bleomycin or UV radiation did not display an increased sensitivity in the mutant background (results not shown). Figure 5.Hypersensitivity of different Arabidopsis mutants to the DNA-damaging agent MMC. Arabidopsis seeds from the mutant lines atbard1-1, atbard1-2, atbrca1-1, atbrca1-2 as well as the double-mutant atbard1-2/atbrca1-1 were tested for their sensitivity to MMC. Wild-type seeds (Columbia) and atku70 (a sensitive control line; Bundock et al, 2002) were used as controls. Seeds were plated on GM medium containing 30 μg MMC/ml, 17 days later seedlings were analysed for their sensitivity. WT: wild type. Download figure Download PowerPoint AtBARD1 is dispensable for meiosis We checked whether the selfed progeny of the T-DNA mutant atbard1 plants was fertile in order to test whether the AtBARD1 protein plays a role during meiosis. Both atbard1 T-DNA insertion mutants produced viable seeds at similar numbers as the wild-type plants. As minor meiotic defects are often correlated with reduced viability of male gametes, pollen of both mutants were analysed with Alexander (1969) staining. However, a similar number of viable pollen could be detected in wild-type and mutant anthers, indicating that AtBARD1 is not necessarily required for the progression of meiosis in plants (data not shown). Intrachromosomal HR is reduced in atbard1 mutant plants and less inducible by genotoxic stress To test the frequency of somatic HR in planta, a well-established recombination assay using the transgenic line 651 was performed (Swoboda et al, 1994). The recombination substrate within the transgene consists of two overlapping fragments of the β-glucuronidase gene (GUS; uidA) interrupted by a hygromycin selectable marker gene. The separated uidA sequences share a common overlap of 566 bp in inverted orientation. HR between the two overlapping DNA sequences produces a functional uidA gene. Cell clusters expressing β-glucuronidase activity can be detected as blue sectors after histochemical staining, and it was shown before that these sectors indeed arise from recombination events (Swoboda et al, 1994). The homozygous atbard1-1 and 1-2 mutants were crossed with a transgenic line carrying the 651 transgene and selfed again to obtain plants homozygous with respect to the atbard1 insertion as well as the 651 transgene. Seedlings were incubated in liquid germination medium (GM) with and without bleomycin (10 μg/ml). Bleomycin is a radiomimeticum causing single-stranded breaks (SSB) and DSB (Harsch et al, 2000). Next, recombination events were counted in 12-day-old seedlings. For both mutant atbard1 lines, the distribution and frequency of recombination events were determined. Figure 6 shows a representative individual experiment for each mutant line. The significance of the differences of the HR events between mutants and segregated control plants was confirmed by the pair-wise nonparametric Mann–Whitney U-test. The experiment was repeated three times for each line (Table I). In all three independent experiments, a significant reduction of HR was found in the mutant backgrounds, either with or without genotoxic stress. A comparison between the untreated segregated control plants and the untreated atbard1-1 and atbard1-2 homozygous plants showed that the frequency of recombination events in the mutant plants was 2–3 times and about 10 times lower, respectively, as compared to the control line (Figure 6A and B; Table I). Figure 6.HR events in 651/atbard1 seedlings. The diagrams show the percentage of seedlings with a given number of blue spots. (A) Untreated atbard1-1, (B) untreated atbard1-2, (C) bleomycin-treated atbard1-1 and (D) bleomycin-treated atbard1-2. atbard1 seedlings are displayed as black bars, segregated control plants homozygous for AtBARD1 are shown as white bars. Download figure Download PowerPoint Table 1. Somatic HR in atbard1-1 and segregated control plants (A), and in atbard1-2 and segregated control plants (B) Control atbard1 Relation n N m1 n N m2 m2/m1 (A) No genotoxic stress 35 16 0.46 34 7 0.21 0.46 36 22 0.61 33 6 0.18 0.30 35 36 1.03 33 11 0.33 0.32 Mean 0.70±0.20 0.24±0.08 0.36* Bleomycin induction (10 μg/ml) 33 2733 77.97 34 803 23.61 0.30 33 1947 61.97 34 393 11.56 0.19 34 2431 57.19 34 342 10.06 0.18 Mean 65.71±10.88 15.08±7.43 0.22* (B) No genotoxic stress 33 16 0.48 34 2 0.06 0.13 31 20 0.65 32 3 0.09 0.14 34 22 0.65 34 1 0.03 0.04 Mean 0.59±0.10 0.06±0.03 0.10* Bleomycin induction (10 μg/ml) 34 1422 41.82 34 28 0.82 0.02 34 3063 90.09 34 65 1.91 0.02 33 2673 81.00 34 84 2.47 0.03 Mean 70.97±25.65 1.73±0.84 0.22* Data are numbers of plants tested (n), total blue stained recombination spots (N), and the mean number of spots per plant per chromosomal recombination assay (m1: control; m2: atbard1) in three different experiments (*calculated from the means of the three experiments). When both mutant atbard1 lines were challenged with bleomycin (10 μg/ml), the frequency of recombination events increased by about two orders of magnitude in the control lines, whereas the induction was significantly lower in both atbard1-1 and atbard1-2 lines (Figure 6C and D; Table I). This is also demonstrated by the fact that in all cases the relation between the mean recombination frequencies of mutant (m2) and segregated control plants (m1) was lower with than without application of genotoxic stress (see Table I last column m2/m1). Taking into account the enhanced sensitivity to MMC of atbard1-2 in comparison to atbard1-1, the differences between the two mutants in HR can be taken as a hint that only in case of atbard1-2 the insertion of the T-DNA into the gene resulted in a 'true' null mutation. Independent of the different degrees of deficiency found in the two mutant lines, our results clearly demonstrate that AtBARD1 is not only required for the repair of DSBs by HR under standard growth conditions, but also for the regulation of HR induction after application of genotoxic stress. AtBRCA1 and AtBARD1 are epistatic for cross-link repair Our two-hybrid data as well as in planta experiments indicated that AtBARD1 and AtBRCA1 physically interact. To demonstrate a genetic interaction we screened the sequence databases of T-DNA insertion mutants on the SIGnAL (Salk Institute Genomic Analysis Laboratory; Alonso et al, 2003) and Garlic (Syngenta) webpages (Sessions et al, 2002). Two atbrca1 T-DNA mutant lines were identified. The insertion sites were determined in detail by PCR. Figure 7 provides a precise characterisation of the T-DNA insertions in the AtBRCA1 gene. The first insertion in the AtBRCA1 gene, SALK 014731, atbrca1-1, is located in the fourth exon. Left borders of T-DNA were found at both ends of the insert, indicating the integration of T-DNAs in tandem inverted orientation. The integration led to the deletion of 18 nucleotides of the fourth exon (Figure 7A and B). The second insertion, GARLIC 916_C09, atbrca1-2, was located in the fifth intron more to the middle of the gene, and the insert is flanked by a right and a left T-DNA border. This insertion led to the del
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