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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.

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Related in: MedlinePlus

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.
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pgen-1000093-g002: 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.

Mentions: Plants compensate the deleterious effects of UV radiation by various mechanisms including the accumulation of secondary metabolites [23]. To check whether the UV-C hypersensitive phenotype of cul4, ddb1a and ddb2 mutant lines is the consequence of a defect in a DNA repair mechanism, cell extracts of WT, cul4-1 and ddb2-2 plants were used in an in vitro DNA repair assay as described in [24]. This assay measures the efficiency of DIG dUTP incorporation in a UV-C damaged plasmid in the presence of plant cell extracts. Thereby the efficiency of dark repair of UV-induced DNA damage can be evaluated. Strikingly, cell extracts derived from the mutant plants were less efficient in DIG dUTP incorporation after 1 and 2 h of incubation (Figure 2). Taken together these results show that the UV hypersensitivity of cul4 and ddb2 (and most likely ddb1a) mutant plants is due to a defect in synthesis-dependent repair of UV-induced DNA lesions, indicating an important role for these factors in the excision repair process.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000093-g002: 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.
Mentions: Plants compensate the deleterious effects of UV radiation by various mechanisms including the accumulation of secondary metabolites [23]. To check whether the UV-C hypersensitive phenotype of cul4, ddb1a and ddb2 mutant lines is the consequence of a defect in a DNA repair mechanism, cell extracts of WT, cul4-1 and ddb2-2 plants were used in an in vitro DNA repair assay as described in [24]. This assay measures the efficiency of DIG dUTP incorporation in a UV-C damaged plasmid in the presence of plant cell extracts. Thereby the efficiency of dark repair of UV-induced DNA damage can be evaluated. Strikingly, cell extracts derived from the mutant plants were less efficient in DIG dUTP incorporation after 1 and 2 h of incubation (Figure 2). Taken together these results show that the UV hypersensitivity of cul4 and ddb2 (and most likely ddb1a) mutant plants is due to a defect in synthesis-dependent repair of UV-induced DNA lesions, indicating an important role for these factors in the excision repair process.

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