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. 2018 Feb 20;176(4):2613–2622. doi: 10.1104/pp.18.00123

The Transcriptional Coactivator ADA2b Recruits a Structural Maintenance Protein to Double-Strand Breaks during DNA Repair in Plants1

Jianbin Lai 1,2,3, Jieming Jiang 1,2, Qian Wu 1, Ning Mao 1, Danlu Han 1, Huan Hu 1, Chengwei Yang 1,3
PMCID: PMC5884601  PMID: 29463775

ADA2b, a transcriptional coactivator, recruits SMC5, a chromosome structural maintenance protein, to double-strand DNA breaks in the plant DNA damage response.

Abstract

DNA damage occurs in all cells and can hinder chromosome stability and cell viability. Structural Maintenance of Chromosomes5/6 (SMC5/6) is a protein complex that functions as an evolutionarily conserved chromosomal ATPase critical for repairing DNA double-strand breaks (DSBs). However, the mechanisms regulating this complex in plants are poorly understood. Here, we identified the transcriptional coactivator ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b) as an interactor of SMC5 in Arabidopsis (Arabidopsis thaliana). ADA2b is a conserved component of the Spt-Ada-Gcn5 acetyltransferase complex, which functions in transcriptional regulation. Characterization of mutant and knockdown Arabidopsis lines showed that disruption of either SMC5 or ADA2b resulted in enhanced DNA damage. Both SMC5 and ADA2b were associated with γ-H2AX, a marker of DSBs, and the recruitment of SMC5 onto DSBs was dependent on ADA2b. In addition, overexpression of SMC5 in the ada2b mutant background stimulated cell death. Collectively, our results show that the interaction between ADA2b and SMC5 mediates DNA repair in plant cells, suggesting a functional association between these conserved proteins and further elucidating mechanisms of DNA damage repair in plants.

DNA damage can considerably destabilize the genome in all cell types. Double-strand breaks (DSBs) can result from errors in DNA replication or exposure to ionizing radiation and genotoxic chemicals. Cells respond to DSBs by controlling the cell cycle, repairing DNA lesions, and activating programmed cell death if the damage is extensive (Hu et al., 2016). When a DSB occurs, histone H2AX is phosphorylated via ATAXIA-TELANGIECTASIA MUTATED (ATM) or ATM/RAD3-RELATED, and other foci-forming factors are recruited to the DSB. DSBs are repaired by homologous recombination or nonhomologous end joining. Homologous recombination is an important mechanism in which intact homologous regions are used as a template for repair. The sequential recombination at DSBs requires the recombinase Rad51 and is completed by new DNA synthesis (Spampinato, 2017). Structural Maintenance of Chromosome (SMC) complexes, including the cohesion, condensing, and SMC5/6 complexes, regulate chromosome architecture and organization (Uhlmann, 2016). The SMC5/6 complex, which plays a critical role in chromosome structure maintenance and homologous recombination in DSB repair, is composed of SMC5, SMC6, and seven non-SMC elements (Wu and Yu, 2012). Similar to in yeast and mammalian cells, the components of the SMC5/6 complex are involved in DNA recombination and repair in plants (Li et al., 2017; Watanabe et al., 2009; Xu et al., 2013; Yan et al., 2013); however, the mechanisms regulating this complex in plants are poorly understood.

Upon DNA damage, chromatin-associated factors may facilitate DSB repair. A previous study in mammalian cells indicated that the SWI/SNF chromatin remodeling complex facilitates the phosphorylation of histone H2AX, and the catalytic subunit of SWI/SNF binds to nucleosomes by interacting with acetylated histone H3 on DSBs (Lee et al., 2010). Subsequently, it was reported that recruitment of the SMC5/6 complex to DSBs is mediated by the BRCT domain-containing protein RTT107 in Saccharomyces cerevisiae (Leung et al., 2011). However, our bioinformatics analysis indicated that Arabidopsis (Arabidopsis thaliana) lacks a RTT107 homolog, implying that SMC5/6 may be recruited by another mechanism in plant cells.

In this study, we examined whether chromatin-associated proteins also are involved in regulating the SMC5/6 complex in plants. Via a yeast two-hybrid screen, we identified ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b, also named PRZ1; Sieberer et al., 2003; Vlachonasios et al., 2003) as a SMC5-interacting protein. In yeast and mammalian cells, ADA2 is a conserved transcriptional activator protein in the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, which is known to regulate gene expression. ADA2 interacts with histone H3 and may recruit GENERAL CONTROL NON-DE-REPRESSIBLE5 (GCN5) onto the chromatin, where it mediates histone acetylation (Wang and Dent, 2014). In Arabidopsis, ADA2b also enhances the histone acetyltransferase activity of GCN5 and is involved in development (Mao et al., 2006; Sieberer et al., 2003; Vlachonasios et al., 2003); however, its involvement in DSB repair has not been reported. Our results reveal a functional association between these conserved proteins and their recruitment to DSBs, thereby providing insight into the molecular mechanisms underlying DNA repair in plant cells.

RESULTS

ADA2b Interacts with SMC5

The SMC5/6 complex is essential for DSB repair in a range of species, but the mechanisms regulating this complex are unclear. Previous studies in our laboratory indicated that AtMMS21, a SUMO ligase that interacts with SMC5, plays a role in the plant DNA damage response (Xu et al., 2013; Yuan et al., 2014). Therefore, we were interested in examining the mechanisms regulating SMC5, a critical subunit of the SMC5/6 complex. Because DNA repair occurs on chromosomes, it is possible that some other protein complexes associated with chromatin also are involved in this process.

Based on this hypothesis, we conducted a yeast two-hybrid screen in which we evaluated the ability of 10 chromatin-associated factors, namely GCN5, ADA2b, SWI3C, BRM, HDA6, HDA19, AS2, FAS2, MSI4, and NRP1, to interact with SMC5. Of these proteins, only ADA2b interacted with SMC5 (Fig. 1A). Previous studies showed that ADA2b interacts with acetyltransferase GCN5 (Mao et al., 2006), whereas SMC5 forms a complex with SMC6 (Wu and Yu, 2012). However, yeast two-hybrid analysis indicated that ADA2b does not interact with SMC6 and that GCN5 does not interact with SMC5 (Supplemental Fig. S1), supporting the notion that ADA2b specifically interacts with SMC5 (Fig. 1A). We confirmed the physical interaction between SMC5 and ADA2b in an in vitro pull-down assay; compared with the GST control, ADA2b-FLAG precipitated with GST-SMC5 (Fig. 1B).

Figure 1.

Figure 1.

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SMC5 interacts with ADA2b. A, The interaction between ADA2b (fused to AD [activation domain]) and SMC5 (fused to BD [DNA-binding domain]) was detected in a yeast two-hybrid assay. B, The interaction between ADA2b and SMC5 was confirmed by an in vitro pull-down assay. ADA2b fused with a FLAG tag was incubated with immobilized GST-SMC5 or GST (control). The precipitated ADA2b was detected by anti-FLAG antibody via immunoblotting. The protein levels of GST-SMC5 and GST are shown in the bottom panel using Coomassie Blue stain. C, Identification of the domain on SMC5 required for its interaction with ADA2b by yeast two-hybrid analysis. The N-terminal region is indicated in red and the C-terminal region in blue. D, Identification of the domain on ADA2b required for its interaction with SMC5 by yeast two-hybrid analysis. The N-terminal region is shown in gray and the C-terminal region in blue. The SANT domain is in pink. E, Colocalization of SMC5-YFP and CFP-ADA2b in protoplasts. Bars = 10 μm. The top panels show the fluorescence images, whereas the bottom panels show the bright-field and merged images. F, The interaction between SMC5 and ADA2b in an in vivo coimmunoprecipitation assay. The 35S:SMC5-YFP or the 35S:YFP construct was coexpressed with 35S:ADA2b-FLAG in leaf protoplasts. Total protein extracts were immunoprecipitated with immobilized anti-GFP agarose. The proteins from lysates (left) and immunoprecipitated samples (right) were detected on immunoblots using anti-YFP or anti-FLAG antibodies. IP, Immunoprecipitation; IB, immunoblot.

To investigate the SMC5/ADA2b interaction in detail, we performed a yeast two-hybrid experiment using a series of truncated proteins. We found that the C-terminal region of SMC5 is essential for its interaction with ADA2b (Fig. 1C), whereas the C-terminal region of ADA2b, which lacks the SANT domain (Sterner et al., 2002), is critical for its interaction with SMC5 (Fig. 1D). Moreover, in a transient expression experiment in Arabidopsis protoplasts, both SMC5-YFP and CFP-ADA2b were localized to the plant nucleus (Fig. 1E), further supporting their interaction. Coimmunoprecipitation experiments using protoplasts confirmed specific SMC5/ADA2b interaction (Fig. 1F). These data show conclusively that SMC5 interacts with ADA2b both in vitro and in vivo.

Disruption of SMC5 or ADA2b Enhances DNA Damage

Given that SMC5 is a key component of the conserved SMC5/6 complex in a variety of species, it is possible that it also is involved in DNA repair in Arabidopsis. Previous studies showed that mutation of SMC5 is embryo lethal (Watanabe et al., 2009; Xu et al., 2013). To better characterize its function in DNA repair, we knocked down the expression of SMC5 via RNA interference in transgenic plants (Supplemental Fig. S2A). Growth of SMC5 RNAi plants was stunted compared with untransformed wild-type plants (Fig. 2A). A RT-qPCR analysis (Fig. 2B) and a comet assay (Fig. 2C; Supplemental Fig. S3A) indicated that the expression levels of genes associated with the DNA damage response, as well as the levels of DNA damage, were significantly increased when SMC5 expression was knocked down. Given that disruption of the DNA repair machinery may increase the sensitivity to DNA damage, we tested the sensitivity of SMC5 RNAi seedlings via treatment with methyl methanesulfonate (MMS), a DSB-inducing reagent (Yuan et al., 2014). SMC5 RNAi plants were more sensitive than the wild type to DNA damage and exhibited lower survival rates (Fig. 2D), thus providing evidence that SMC5 functions in DNA repair.

Figure 2.

Figure 2.

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Disruption of SMC5 or ADA2b enhances DNA damage. A and E, The developmental phenotype of 3-week-old SMC5 RNAi (A) and ada2b (E) Arabidopsis plants. Bars = 1 cm. B and F, Expression levels of genes associated with DNA damage in the rosette leaves of 3-week-old SMC5 RNAi (B) and ada2b (F) plants. The RT-qPCR data are means ± se from triplicate experiments. ***P < 0.001, **P < 0.01, *P < 0.05, Student’s t test. C and G, The DNA damage status of SMC5 RNAi (C) and ada2b (G) plants observed via a comet assay. The results are representative of three independent biological experiments. Bars = 50 μm. D and H, Representative images of the MMS sensitivity of the SMC5 RNAi (D) or ada2b (H) plants. The 5-d-old seedlings were transferred to Murashige and Skoog medium with or without 50 μg mL−1 MMS. Photographs were taken 7 d after transfer. The survival rates are means ± sd from three independent experiments. ***P < 0.001, Student’s t test. WT, Wild type.

Given that ADA2b interacts with SMC5, it is possible that ADA2b also is involved in DNA damage repair in plant cells. In yeast and Drosophila cells, ADA2b homologs have been reported to be involved in the response to DNA damage (Qi et al., 2004), but the precise mechanism is unclear. Previous studies showed that mutation of ADA2b affects Arabidopsis development (Fig. 2E; Kornet and Scheres, 2009; Sieberer et al., 2003; Vlachonasios et al., 2003), but there is no evidence for its role in the DNA damage response. A RT-qPCR analysis indicated that the expression of several DNA damage-associated genes was dramatically increased in ada2b plants (Fig. 2F). Consistently, we observed increased DNA damage in the absence of ADA2b under normal conditions via the comet assay (Fig. 2G; Supplemental Fig. S3B). To measure the sensitivity of the ada2b mutants to DNA damage, we monitored their growth on MMS-containing medium. Most ada2b seedlings perished upon treatment with MMS (Fig. 2H), suggesting the importance of ADA2b in DNA repair.

Both SMC5 and ADA2B Are Localized to DSBs in Damaged DNA

When DNA damage occurs, H2AX is phosphorylated at DSBs and mediates the recruitment of other foci-forming factors (Dantuma and van Attikum, 2016; Polo, 2015). In mammalian cells, experimental evidence indicated that the SMC5/6 complex is recruited to DSBs to maintain the DNA structure during repair (Potts et al., 2006). In this study, we found that when SMC5-YFP was expressed in plant cells, the fluorescence signal was detected in the nucleus (Fig. 1E). This raised the question as to whether the localization of SMC5 would change during DNA damage in plant cells. Given that phosphorylation of H2AX specifically occurs on DSBs, the antibody recognizing γ-H2AX (the phosphorylated H2AX; Lorković et al., 2017) was used to label the DSB regions. Under normal conditions, no γ-H2AX signal was detected; however, when DNA damage was induced by MMS, some γ-H2AX foci were observed in the nucleus, indicating the position of damage on genomic DNA (Fig. 3A). Interestingly, fluorescent signals from SMC5-YFP also were concentrated onto the foci, almost overlapping with γ-H2AX signals (Fig. 3A), suggesting that SMC5 is recruited to DSBs during DNA damage.

Figure 3.

Figure 3.

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ADA2b is critical for the recruitment of SMC5 to DSBs during DNA damage. A and B, Colocalization of SMC5-YFP (A) or YFP-ADA2b (B) with γ-H2AX during DNA damage. 35S:SMC5-YFP or 35S:YFP-ADA2b was expressed in protoplasts incubated in W5 medium with or without MMS overnight, after which cells were fixed for immunostaining with anti-γ-H2AX antibody (labeled by Alexa Fluor 555). The YFP, Alexa Fluor 555, and DAPI signals were detected by confocal microscopy. C and D, Colocalization of SMC5-YFP (C) or YFP-ADA2b (D) with Rad51C-CFP under MMS treatment. E, Colocalization of SMC5-YFP and CFP-ADA2b under MMS treatment. F and G, Representative localization of YFP-ADA2b in wild-type (F) or SMC5 RNAi (G) protoplasts with or without MMS treatment. H and I, Representative localization of SMC5-YFP in wild-type (H) or ada2b-3 mutant (I) protoplasts with or without MMS treatment. Histogram data (F–I) for the percentage of cells with (in purple) or without (in blue) DSB foci from three independent experiments are means ± sd. J, Localization of the C-terminal region of ADA2b under MMS treatment. K, Localization of SMC5-YFP under MMS treatment in the presence of the C-terminal region of ADA2b. Bars = 5 μm. All experiments described in this figure were performed at least three times with similar results. WT, Wild type.

To determine whether ADA2b also associates with DSBs, we next examined the localization of YFP-ADA2b in cells with DNA damage. The localization of YFP-ADA2b also overlapped with γ-H2AX in the presence of MMS (Fig. 3B). Given that Rad51 proteins are critical components in DNA repair (Biedermann et al., 2017; Liu et al., 2017), Rad51C-CFP fusion proteins also were used as a marker for DSBs. Both SMC5-YFP and YFP-ADA2b colocalized with the Rad51C-CFP foci under MMS treatment (Fig. 3, C and D), confirming the results observed using γ-H2AX. Interestingly, the subcellular localization of ADA2a, a homolog of ADA2b in Arabidopsis, was not responsive to MMS (Supplemental Fig. S4), suggesting a specific role of ADA2b in this process. In addition, the colocalization of SMC5 and ADA2b at damaged DNA was confirmed following their coexpression with different fluorescence proteins (Fig. 3E). Taken together, our data suggest that SMC5 and ADA2b are recruited to DSBs when DNA damage occurs in plant cells.

ADA2b Is Essential for the Recruitment of SMC5 onto DSBs

To understand the recruitment mechanism of SMC5/ADA2b onto DSBs, we performed several experiments to detect whether the localization of SMC5 and ADA2b to damaged DNA was mutually dependent. First, the localization of YFP-ADA2b was observed in wild-type and SMC5 RNAi cells with or without MMS treatment. Under normal conditions, YFP-ADA2b was generally localized in the nucleus in both cell genotypes (Fig. 3, F and G). In the presence of MMS, the localization of YFP-ADA2b was concentrated at foci in wild-type and SMC5 RNAi cells (Fig. 3, F and G), suggesting that the localization of ADA2b to DSBs is not dependent on SMC5.

Similarly, we monitored SMC5-YFP localization in wild-type and ada2b cells with or without MMS treatment. In the absence of DNA damage stress, SMC5-YFP was generally distributed in the nucleus in both cell genotypes (Fig. 3, H and I). When the cells were incubated with MMS, the SMC5-YFP foci were observed in most wild-type cells (Fig. 3H) but not in around 80% of ada2b cells (Fig. 3I), suggesting that the localization of SMC5-YFP to DSBs is predominantly dependent on ADA2b. Further analysis showed that SMC6, another conserved subunit of the SMC5/6 complex, was colocalized with SMC5 in the foci under MMS treatment, and the recruitment of SMC6 also was dependent on ADA2b (Supplemental Fig. S5).

Because the SANT motif in the N-terminal region of ADA2b is a domain known to interact with histones (Boyer et al., 2004; Sterner et al., 2002), we performed a similar pull-down assay and found that the N-terminal region of Arabidopsis ADA2b also strongly interacted with histone H3 in vitro (Supplemental Fig. S6A), implying a conserved function of this motif among species. However, when the N terminus of ADA2b was fused with YFP and expressed in plant cells, the truncated protein localized throughout the whole cell and was not responsive to MMS treatment (Supplemental Fig. S6B), whereas the C terminus of ADA2b was localized in the nucleus but did not form foci in the presence of MMS (Fig. 3J). In the presence of MMS, when SMC5-YFP was coexpressed with the N-terminal or C-terminal region of ADA2b, the foci formation of SMC5-YFP was blocked by the C terminus (Fig. 3K) but not the N terminus of ADA2b (Supplemental Fig. S6C), consistent with our analysis of interacting regions from yeast two-hybrid assays. Therefore, the N terminus of ADA2b binds with histones and its C terminus interacts with SMC5, but both regions of ADA2b are necessary for its DSB recruitment.

Overexpression of SMC5 Enhances Cell Death in ada2b Plants

Our current evidence indicated that the localization of SMC5 to DSBs is dependent on ADA2b; when ADA2b is nonfunctional, SMC5 distributes abnormally during DNA damage. Therefore, we investigated the effect of excess SMC5 proteins in the absence of ADA2b in plants. We overexpressed SMC5 in wild-type plants (Supplemental Fig. S2B) and crossed these plants with ada2b plants to generate homozygous lines. Compared with the wild type, 5-week-old ada2b plants displayed mild leaf senescence, possibly resulting from accumulation of DNA damage. Surprisingly, overexpression of SMC5 in ada2b plants dramatically increased leaf senescence (Fig. 4A). Results of Trypan blue staining confirmed that the level of cell death was increased when SMC5 was overexpressed in ada2b plants but not in wild-type plants (Fig. 4B), implying that these proteins are functionally associated in plant cells.

Figure 4.

Figure 4.

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Overexpression of SMC5 enhances cell death in ada2b plants. A, Phenotypes of the rosette leaves of 5-week-old ada2b-3 plants and two independent SMC5-overexpressing lines in the ada2b-3 background. Bar = 1 cm. B, Cell death in the leaves depicted in A was quantified by Trypan blue staining. Bars = 200 μm. C, A proposed model for the recruitment of SMC5 onto DSBs via ADA2b in plant cells. WT, Wild type.

DISCUSSION

The SMC5/6 complex is essential for DNA repair at DSBs in various organisms (Uhlmann, 2016). In this study, we provide direct evidence that SMC5 functions in DNA repair in plant cells. To fully understand the DSB repair mechanism, it is important to investigate how the SMC5/6 complex is specifically localized to DSBs. Recruitment of the SMC5/6 complex is mediated by RTT107 in yeast cells (Hang and Zhao, 2016; Leung et al., 2011); however, as there is no RTT107 homolog in Arabidopsis, a different mechanism must operate in plant cells. In mammalian cells, several chromatin-associated factors, including histone modification enzymes and chromatin remodeling components, mediate the structural changes to chromosomes that provide sequential access of the repair mechanism to DSBs (Lee et al., 2010), thereby providing a possible alternative route for SMC5/6 recruitment. However, it is unclear whether these functional chromatin-related factors associate directly with SMC5/6, and thus, it is unknown whether SMC5/6 localization is the result of chromosome change or is mediated via interaction with chromatin-related factors. In this study, we identified the transcriptional adaptor ADA2b as a SMC5-interacting protein in plant cells.

ADA2b is a conserved adaptor protein that connects histones with the acetylase GCN5 (Gamper et al., 2009). Although ADA2b has been reported to be associated with the DNA damage response in yeast and Drosophila (Qi et al., 2004), its precise role in this process is unknown. The results in this study show that SMC5 and ADA2b associate with γ-H2AX foci under DNA damage stress and that the recruitment of SMC5 to DSBs requires ADA2b. Our data from in vitro pull-down analyses supported that the N terminus of Arabidopsis ADA2b is essential for its interaction with histones, consistent with the conclusions made in other species (Boyer et al., 2004; Sterner et al., 2002), whereas the C terminus of ADA2b interacts with SMC5 in the yeast two-hybrid system, and expression of this C-terminal region disrupts SMC5 localization to DSBs. However, the results from the localization of truncated ADA2b indicated that both regions are necessary for correct recruitment of ADA2b to DSBs. Therefore, the N terminus of ADA2b may bind with histone proteins, while the C-terminal region of ADA2b associates with SMC5, but an additional region on ADA2b also may contribute to its recruitment to DSBs.

In ada2b, SMC5 fails to be recruited to DSBs and is mislocalized in the nucleus, possibly enhancing DNA damage. Given that the SMC5/6 complex consists of multiple components, the mislocalization of SMC5 may disrupt the function of the complex. Results from yeast two-hybrid analysis indicated that SMC6 does not interact with ADA2b, and our data also showed that the DSB localization of both SMC5 and SMC6 requires ADA2b; therefore, the correct localization of SMC6, or even the whole complex, may be dependent on the recruitment of SMC5 mediated by ADA2b. Because of its importance in the SMC5/6 complex, excess amount of SMC5 may interfere with the balance between different components. Interestingly, overexpression of SMC5 in the wild-type background has no effect on development, but SMC5 overexpression enhances cell death in the ada2b mutant. This dosage effect suggests a functional association between SMC5/ADA2b and may be due to an imbalance of the complexes.

We propose a model whereby upon DNA damage, H2AX is phosphorylated at DSBs, then ADA2b specifically binds to the histones in this region and mediates subsequent localization of SMC5 for further DNA repair (Fig. 4C). Some interesting questions remain for further investigation. For instance, what does the additional region on ADA2b contribute to its DSB recruitment? Are other factors also required for correct recruitment of the SMC5/6 complex in plant cells? Is ADA2b-mediated histone acetylation also important in this process? Finally, does the interaction between SMC5 and ADA2b affect assembly of the SAGA and SMC5/6 complexes? Given that SMC5 and ADA2b are conserved factors but their association has not been reported in other species, it would be interesting to determine whether this mechanism is conserved in other plants or even in mammalian cells.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Growth conditions used for Arabidopsis (Arabidopsis thaliana) Columbia-0 (wild type), ada2b-1 (prz1-1; CS9574, Sieberer et al., 2003), and ada2b-3 (SALK_019407, Kornet and Scheres, 2009) have been described previously. Seeds were surface sterilized for 2 min in 75% ethanol followed by 5 min in 1% NaClO solution, rinsed five times with sterile water, plated on Murashige and Skoog (MS) medium with 1.5% Suc and 0.8% agar, and then stratified at 4°C in darkness for 2 d. Plants were grown under long-day conditions (16 h light/8 h dark) at 22°C. For MMS treatment, 5-d-old seedlings on Murashige and Skoog medium were transferred to Murashige and Skoog medium with or without 50 μg mL−1 MMS (Sigma; catalog no. 129925) for 7 d before being scored and photographed.

Yeast Two-Hybrid Assays

Yeast two-hybrid assays were performed according to the manufacturer’s instructions for the Matchmaker GAL4-based Two-Hybrid System 3 (Clontech). The coding sequence (CDS) of SMC5 was cloned into the pGBKT7 vector. The CDSs of ADA2b were cloned into the pGADT7 vector. To characterize the interaction domain, the truncated products were generated as described in the figure legends. Protein interactions were tested by stringent (SD/−Leu/−Trp/−His) selection supplied with 3-amino-1,2,4-triazole followed by β-Galactosidase activity measurement (Clontech). The information of primers used in this study is listed in Supplemental Table S1.

In Vitro Pull-Down Assay

The CDS of SMC5 was cloned into pGEX4T-1, whereas the CDS of ADA2b fused with a FLAG tag was cloned into pET28A. All proteins were expressed in BL21. GST or GST-SMC5 recombinant proteins were incubated with GST resins (GE Healthcare) in a binding buffer (50 mm Tris, pH 7.4, 120 mm NaCl, 5% glycerol, 0.5% Nonidet P-40, 1 mm PMSF, 1 mm β-mercaptoethanol) for 2 h at 4°C, collected, and mixed with supernatant containing His6-ADA2b-FLAG, and then incubated at room temperature for 60 min. After rinsing five times with washing buffer (50 mm Tris, pH 7.4, 120 mm NaCl, 5% glycerol, 0.5% Nonidet P-40), the bound proteins were boiled in SDS sample buffer and subjected to SDS-PAGE and immunoblotting using anti-FLAG antibody (Sigma).

Coimmunoprecipitation

The 35S:SMC5-YFP plasmid was constructed by fusing YFP with the CDS of SMC5 in the pSAT6-EYFP-N1 vector with a 35S promoter. The 35S:ADA2b-FLAG plasmid was generated by fusing a FLAG tag with the genomic region of ADA2b under a 35S promoter in a pBluescript-based vector (Liu et al., 2016). Using YFP alone as a control, the plasmids were cotransformed into Arabidopsis leaf protoplasts (Yoo et al., 2007). Forty-eight hours after transformation, the protoplasts were collected for coimmunoprecipitation. Proteins were extracted in extraction buffer (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 10% glycerol, 0.5% Nonidet P-40) containing Protease Inhibitor Cocktail (Roche). After centrifugation at 13,000g for 10 min, the supernatant was incubated with GFP-Trap resin at 4°C for 3 h. The beads were then centrifuged and washed three times with washing buffer (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 10% glycerol). Proteins were eluted with SDS sample buffer and analyzed by immunoblotting using anti-GFP (Abcam) or anti-FLAG (Sigma) antibodies.

Comet Assay

Protoplasts from 3-week-old seedlings were used to perform the comet assay using the Comet Assay Kit from Trevigen (catalog no. 4250-050-K). Comets were stained with SYBR Gold from Life Technologies, captured using a Zeiss LSM 710 confocal microscope with excitation/emission wavelengths of 488 nm/505 to 530 nm, and analyzed by CASP Comet Assay Software Project.

Fluorescence Microscopy

To analyze the localization of ADA2b and SMC5, the genomic DNA of ADA2b was cloned into the 35S:CFP/YFP vector, which is based on pBluescript, and the CDS of SMC5 was cloned into the pSAT6-EYFP-N1 vector. Protoplast transformation was performed as described previously (Yoo et al., 2007). After transformation, 3-week-old Arabidopsis protoplasts were incubated in W5 medium with or without 15 μg mL−1 MMS (Sigma; catalog no. 129925) overnight. For those transformations with YFP signal only, DAPI (Sigma; catalog no. D9542) was used as a marker for the nucleus. The signals were examined by confocal microscopy (Leica LSM800).

Immunofluorescence Staining

The SMC5-YFP/YFP-ADA2b construct was transferred into protoplasts generated from leaves of 2- to 3-week-old seedlings and incubated in W5 medium with or without 15 μg mL−1 MMS (Sigma; catalog no. 129925) overnight. The immunofluorescence assay was performed as described previously (Bowler et al., 2004) with slight modifications. Approximately 100 μL of Poly-l-Lys solution (Sigma; catalog no. P8920) was added to a special cell culture dish (NEST; catalog no. 801002) and air dried at 65°C. Approximately 200 μL of protoplasts was placed in the dish and settled for approximately 1 h. Cells were fixed in 200 to 300 μL of 2% paraformaldehyde in PHEM buffer for 10 min at room temperature, and then postfixed in a solution of methanol:acetone (1:1) at −20°C for 10 min. The protoplasts were washed with PBS containing Nonidet P-40 three times (5 min each) at room temperature. Then, 200 μL of 1% BSA in PBS was added to the dish, and samples were incubated for 30 min at 37°C. Subsequently, the primary antibody against γ-H2AX (Lorković et al., 2017) was used at a 1:200 dilution at 4°C overnight. The Alexa Fluor 555-coupled goat antirabbit antibody (Bioss; catalog no. bs-0295G-AF555) was used as the fluorescently labeled secondary antibody at a 1:400 dilution and incubated for 45 min at 37°C. The samples were stained with DAPI (Sigma; catalog no. D9542), and the signals were captured using a Zeiss LSM 710 confocal microscope with the following excitation/emission wavelengths: 514/513 to 542 nm for YFP, 405/410 to 488 nm for DAPI, and 543/555 to 697 nm for Alexa Fluor 555.

Gene Expression Analysis

For analysis of gene expression, all the rosette leaves from 3-week-old plants (five plants for each replicate, the replicates were from plants grown in different pots at the same time; at least three independent experiments from plants grown at different times showed similar results) were collected for RNA extraction. Total RNA was extracted using the Plant RNAprep Pure Kit (Magen) with DNase I treatment following the manufacturer’s instructions. RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara) and subjected to RT-qPCR. The cDNA template was subjected to PCR using SYBR Premix Ex Taq (Takara) in a Bio-Rad CFX 96 system (C1000 Thermal Cycler) and detected by Bio-Rad CFX Manager software (Bio-Rad).

Generation of Transgenic Plants

For the 35S:SMC5-RNAi construct, the sequence of SMC5 was analyzed to choose a sequence-specific fragment (251 bp) for interference. The forward and reverse forms of the fragment were cloned into the pYLRNAi.5 vector with an intron. The complete interference fragment was transferred into the pFGC5941 vector for plant transformation.

For the 35S:SMC5 overexpressing construct, the CDS of SMC5 was obtained by PCR amplification and cloned into the PBA002-MYC vector. The constructs were transformed into Agrobacterium EHA105, which was then used to transform Arabidopsis (Columbia) by the floral-dip method (Clough and Bent, 1998). To overexpress SMC5 in ada2b plants, the 35S:MYC-SMC5 plants were crossed with ada2b-3. The homozygous offspring were used for phenotypic analysis. Cell death was visualized in detached leaves using lactophenol Trypan blue staining and saturated chloral hydrate destaining (Koch and Slusarenko, 1990).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: ADA2b (At4g16420), SMC5 (At5g15920), GCN5 (At3g54610), SMC6B (At5g61460), and Rad51C (At2g45280), ADA2a (At3G07740).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank the Arabidopsis Biological Resource Center for the seeds used in this study and Professor Frederic Berger for the γ-H2AX antibody. The authors do not have any conflict of interests.

Footnotes

1

This work was supported by the National Natural Science Foundation of China (31670286, 31771504, and 31400314), the University Innovation Program from the Department of Education of Guangdong Province (2014), Guangdong Science and Technology Department (2015A020209155 and 2016A020208014), Guangdong YangFan Innovative and Entepreneurial Research Team Project (2015YT02H032), and Guangzhou Scientific and Technological Program (201607010377).

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