Limits...
Regulation and role of Arabidopsis CUL4-DDB1A-DDB2 in maintaining genome integrity upon UV stress.

Molinier J, Lechner E, Dumbliauskas E, Genschik P - PLoS Genet. (2008)

Bottom Line: Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure.Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR).Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS (UPR2357), conventionné avec l'Université Louis Pasteur, Strasbourg, France.

ABSTRACT
Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2396500&req=5

pgen-1000093-g005: 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).

Mentions: Next, we investigated whether Arabidopsis CUL4, DDB1A and DDB2 proteins form a complex in planta. As no antibody against DDB1A was available and because we further aimed to determine the subcellular localisation of DDB1A and DDB2, we first produced Arabidopsis transgenic plants expressing GFP-tagged versions of both proteins (Figure 5A). To test whether the DDB1A-GFP fusion protein was functional, complementation of the UV-C sensitivity of ddb1a-2 plants was analysed using the root growth assay. Expression of both untagged DDB1A and DDB1A-GFP fusion protein complement the ddb1a-2 UV-C sensitivity compared to the control plants to the same extent (Figure 5B). Therefore DBB1A-GFP expressing plants are suitable for further analyses. Pair-wise immunoprecipitation experiments were conducted using either anti-CUL4 or anti-GFP antibodies. Indeed we found that both DDB1A and DDB2 co-immunoprecipitate with CUL4 (Figure 5C). In addition, we showed an enrichment of DDB2 when co-immunoprecipitated with CUL4 upon UV-C exposure compared to untreated plants (Figure 5D).


Regulation and role of Arabidopsis CUL4-DDB1A-DDB2 in maintaining genome integrity upon UV stress.

Molinier J, Lechner E, Dumbliauskas E, Genschik P - PLoS Genet. (2008)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2396500&req=5

pgen-1000093-g005: 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).
Mentions: Next, we investigated whether Arabidopsis CUL4, DDB1A and DDB2 proteins form a complex in planta. As no antibody against DDB1A was available and because we further aimed to determine the subcellular localisation of DDB1A and DDB2, we first produced Arabidopsis transgenic plants expressing GFP-tagged versions of both proteins (Figure 5A). To test whether the DDB1A-GFP fusion protein was functional, complementation of the UV-C sensitivity of ddb1a-2 plants was analysed using the root growth assay. Expression of both untagged DDB1A and DDB1A-GFP fusion protein complement the ddb1a-2 UV-C sensitivity compared to the control plants to the same extent (Figure 5B). Therefore DBB1A-GFP expressing plants are suitable for further analyses. Pair-wise immunoprecipitation experiments were conducted using either anti-CUL4 or anti-GFP antibodies. Indeed we found that both DDB1A and DDB2 co-immunoprecipitate with CUL4 (Figure 5C). In addition, we showed an enrichment of DDB2 when co-immunoprecipitated with CUL4 upon UV-C exposure compared to untreated plants (Figure 5D).

Bottom Line: Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure.Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR).Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS (UPR2357), conventionné avec l'Université Louis Pasteur, Strasbourg, France.

ABSTRACT
Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

Show MeSH
Related in: MedlinePlus