Figure 1.
cul4, ddb1a and ddb2 insertion mutant lines exhibit hypersensitivity to UV-C.
(A) Genomic organization of the DDB1A and DDB2 loci. T-DNAs are inserted in the 10th exon (black boxes) in the ddb1a-1 and in the 5th intron (black line) in ddb1a-2 mutant plants. Primers a–b, c–d and LBb1 were used for genotyping ddb1a-1 and ddb1a-2, respectively. In the ddb2-2, the transposon is inserted in the 10th exon (black boxes). Primers h–g and Ds5-2a were used for genotyping (see supplemental material for details). (B) Molecular characterisation of DDB1A- and DDB2-mutant plants. RT-PCR analyses were performed for DDB1A expression in WT and the insertional mutants ddb1a-1 and ddb1a-2 using the specific e–f primers. RPL4 primers were used as a control (See supplemental material for details). Immuno-detection of DDB2 protein in two different WT Arabidopsis thaliana ecotypes (Columbia: Col0 and Nossen: No) and in the ddb2-2 insertional mutant. Coomassie blue staining was used as loading control. (C) Root-growth assay. One-week-old mutant (cul4-1, ddb1a-1, ddb1a-2, ddb2-2), and WT control plants were exposed to 600 J/m2 of UV-C. Root growth was measured 24h following irradiation. Root growth was calculated relative to the corresponding untreated plants (±SEM). Eight plants per replicate were used and experiments were performed in triplicates. For all mutants p<0.05, compared to WT plants (both ecotypes).
Figure 2.
In vitro synthesis DNA repair assays of UV-C damaged plasmid.
Cell extracts (20 µg) from WT (Col/Nossen), cul4-1 and ddb2-2 plants were incubated with UV-C damaged (UV-C treated pGEX: +UV-C) and control (untreated pBKS: −UV-C) plasmids in the presence of DIG-dUTP. Incorporation was evaluated during a time course. These pictures are representative of 2 independent experiments.
Figure 3.
Genetic interactions in NER and HR.
One-week-old single and double mutant plants (A) ddb1a-2-ddb2-2; cul4-1-cen2-2; ddb1a-2-cen2-2; ddb2-2-cen2-2 (B) cul4-1-rad10; ddb1a-2-rad10; ddb2-2-rad10; cen2-2-rad1 were exposed to 600 J/m2 of UV-C. Root growth was measured 24h following irradiation. Root growth was calculated relative to the corresponding untreated plants (±SEM). Eight plants per replicate were used and experiments were triplicated. For all the single and double mutants p<0.05, compared to WT plants. Because ddb2-2 is in a different Arabidopsis ecotype, the single control mutants were selected as segregants from each double mutant involving ddb2-2 and are indicated by *.
Figure 4.
Genetic interactions between CUL4, DDB1A, DDB2 and UVR2.
Ten-day-old single (cul4-1, ddb1a-2, ddb2-2, phrI) and double mutant plants (cul4-1 phrI, ddb1a-2 phrI, ddb2-2 phrI) were irradiated with UV-C (3000 J/m2) 3 times in row every 2 days. (A) Graph representing the average number of leaves. Counting was one week after the last UV-C exposure. At least 20 plants were used per replicate and this experiment was duplicated. T-test *p<0.05, compared to WT plants. (B) Graph representing the percentage of bleached plants. Counting was 2 weeks after the last UV-C exposure. At least 20 plants were used per replicate and this experiment was duplicated. (C) Pictures showing the phenotype of plants 14 days after the last UV-C exposure (Bar = 1 cm).
Figure 5.
DDB1A and DDB2 interaction with CUL4 and their subcellular localisation.
(A) Schematic representation of the constructs (mas: mannopine synthase; GFP: Green Fluorescent Protein; T: nos terminator). (B) Root growth assays showing complementation of ddb1a-2 with either DDB1A or DDB1A-GFP ectopically expressed proteins. (C) In vivo pull down of CUL4 with DDB1A and DDB2 proteins. WT, pOEX4GFP-DDB2 and ddb1a-2 pOEX2DDB1A-GFP plants were used for immunoprecipitation assays using either anti-CUL4 (Bernhardt et al., 2006) or anti-GFP antibodies. WT plants were used as controls. (D) In vivo pull down of CUL4 complex upon UV-C exposure. WT plants were used for immunoprecipitation assays using anti-CUL4 antibody. CUL4 and DDB2 were detected before (−) and 15 min upon UV-C exposure (+). (E) Localisation of GFP-DDB2 fusion protein in root cells using confocal microscopy (Bar = 50 µm) and immunolocalisation in root cells (Bar = 5 µm). (F) Localisation of DDB1A-GFP fusion protein in root cells using confocal microscopy (Bar = 25 µm). All pictures are representative of 3 different experiments using independent transgenic lines. Chromatin is stained by DAPI (blue).
Figure 6.
ATR-dependent DDB1A-GFP nuclear shuttling and DDB2 stability.
(A) DDB1A-GFP dynamics upon UV-C exposure (600 J/m2) in WT (upper panel) and in atr-2 mutant plant (lower panel). Bar = 75 µm. Pictures are representative of 3 different experiments using 2 independent transgenic lines. (B) Higher magnification of DDB1A-GFP localisation 15 min upon UV-C exposure in either ddb1a-2 or atr-2 mutant backgrounds. Chromatin is stained by DAPI (blue). (C) Immunoblot revealing DDB2 content upon UV-C exposure (900 J/m2) in WT, cul4-1, ddb1a-2 and atr-2 Arabidopsis plants. Coomassie blue staining was used as loading control.
Figure 7.
Schematic representation of DNA repair pathways in plant cells and control of DDB2 stability upon UV exposure.
As a consequence of UV irradiation, different types of DNA damage are produced such as cyclobutyl pyrimidine dimers (CPDs), (6-4) photoproducts (6-4) Pps, Inter/Intra Cross Link (ICL), 8-oxoG or even DNA double-strand breaks (DSBs). In order to repair these lesions different repair processes including Direct Repair, Homologous Recombination (HR) and Global Genome Repair (GGR) are activated. The GGR pathway involves the CUL4-DDB1A ubiquitin E3 ligase complex. Based on our data, we propose the following model: Under normal growth conditions DDB1A localises predominantly to the cytoplasm, whereas CUL4 and DDB2 are both located into the nucleus. Upon UV irradiation, DDB2 recognises bulky DNA lesions and DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner (direct or indirect). This allows DDB2 degradation by the 26S proteasome and most likely permits subsequent steps of the excision repair process to occur efficiently.