The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1 - PubMed (original) (raw)

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

Tanya Stoyanova et al. Mol Cell Biol. 2008 Jan.

Abstract

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21(Waf1/Cip1). We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53(S18P)) and targets it for degradation in low-dose-UV-irradiated cells. DDB2(-/-) mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53(S18P). Accumulation of p53(S18P) in DDB2(-/-) MEFs causes higher expression p21(Waf1/Cip1). We show that the increased expression of p21(Waf1/Cip1) is the cause NER deficiency in DDB2(-/-) cells because deletion or knockdown of p21(Waf1/Cip1) reverses their NER-deficient phenotype. p21(Waf1/Cip1) was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21(Waf1/Cip1) was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21(Waf1/Cip1) to the NER activity of DDB2.

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Figures

FIG. 1.

DDB2−/− MEFs are deficient in NER as measured by UDS. UDS was measured in wild-type (WT) and DDB2−/− MEFs after UV irradiation as previously described (47). The slides were developed for a week. After development, the numbers of grains per nucleus were obtained by counting 25 non-S-phase nuclei per sample. Numbers of grains per nucleus are plotted in the left panels, and autoradiographs of the grains in nuclei are shown in the right panels.

FIG. 2.

DDB2−/− MEFs are unresponsive to UV-induced nuclear accumulation of DDB1. MEFs from wild-type (WT) or DDB2−/− embryos were grown on coverslips and infected with adenovirus expressing T7-tagged DDB1. Sixteen hours following infection, the cells were treated with UV irradiation (20 J/m2) or IR (5 Gy). One hour after irradiation, the cells were fixed and subjected to immunostaining for T7-tagged DDB1 as described in Materials and Methods. We analyzed the localization of DDB1 in 50 to 60 cells, and a quantification of the immunostaining data is shown in panel d.

FIG. 3.

DDB2-deficient cells are impaired in the proteolysis of p53S18P. MEFs were subjected to UV irradiation (12 J/m2) and then maintained in the medium for the indicated time periods. Extracts (100 μg) were subjected to Western blot assays. The blots were probed with antibodies against p53S18P, p53, DDB1, and Cdk2 or tubulin. (b) MEFs were treated with MG132 and then irradiated with UV. The UV-irradiated MEFs were maintained in medium for the indicated time periods. Extracts (100 μg) were subjected to Western blot analysis. NT, not treated. (c) MEFs were treated with UV irradiation, and 3 h following irradiation, cycloheximide (chx) was added to the culture medium and the cells were harvested at the indicated time points. The extracts (100 μg) were analyzed by Western blot assays. (d) A quantification of phospho-p53 band intensity is plotted against time after cycloheximide addition. The gray line represents the decay in DDB2−/− MEFs, and the black line represents the decay in wild-type MEFs.

FIG. 4.

p53 and p53S18P are unstable specifically in low-dose-UV-irradiated cells. (a) MEFs from wild-type embryos were treated with a low dose (12 J/m2) or a high dose (60 J/m2) of UV irradiation and then maintained in the medium for the indicated time period. Extracts (100 μg) of the cells were analyzed for p53 by Western blot assays. (b) MEFs from wild-type or DDB2−/− embryos were treated with IR (5 Gy). After 3 h, cells were treated with cycloheximide (chx) and harvested at the indicated time points. Extracts (100 μg) were analyzed for p53S18P and p53 by Western blot assays.

FIG. 5.

DDB2 deficiency impedes the ubiquitination of p53S18P. HeLa cells expressing DDB2 shRNA or no shRNA (pSuper) were transfected with plasmids expressing Flag-tagged p53 and six-His-tagged ubiquitin (Ub). The transfected cells were treated also with UV irradiation (12 J/m2) and MG132 (for 5 h). Aliquots (20% of the total) of the transfected cells were analyzed for levels of p53 transgene expression with Flag-tagged antibody and for DDB2 expression with DDB2 antibody (left panel). The remaining aliquots of transfected or untransfected cells were lysed in buffer containing 6 M guanidinium-HCl as previously described (53). The lysates were subjected to Ni-NTA-agarose binding and purification of the ubiquitinated proteins as described in reference . The Ni-NTA-agarose-purified fractions were analyzed for ubiquitinated p53S18P and p53 with specific antibodies in Western blot assays. PolyUb, polyubiquitinated.

FIG. 6.

DDB1 preferentially binds p53S18P. (a and b) MEFs from wild-type (WT) embryos were infected with adenovirus expressing T7 epitope-tagged DDB1 or DDB2. Thirteen hours following infection, the cells were treated with MG132 for 2 h and then irradiated with UV (12 J/m2). Subsequently, the cells were maintained in medium containing MG132 for an additional 3 h. Following that, the cells were harvested and extracts (1.5 mg for the experiments in panel a and 3 mg for the experiments in panel b) of these cells were subjected to immunoprecipitation (IP) with T7 antibody or with isotype-matched immunoglobulin G (IgG). The immunoprecipitates were analyzed for the presence of p53S18P (a) or total p53 (b) by Western blot assay. Total extracts (0.1 mg) were also analyzed for the levels of T7-DDB1, T7-DDB2, p53S18P, and total p53. (c) MEFs were UV irradiated in the presence of MG132. Three hours following irradiation, cells were harvested and extracts (2 mg) were subjected to immunoprecipitation with a DDB1 antibody or a control (Cont) antibody. The immunoprecipitates were subjected to Western blot assays with the phopsho-p53 antibody. In this experiment, the blot corresponding to the immunoprecipitate lanes was developed with Fc-horseradish peroxidase (Pierce) instead of a secondary antibody to avoid signals from IgG.

FIG. 7.

Cul4A-DDB1 accelerates the decay of p53S18P. DDB2−/− MEFs were infected with Cul4A expression adenovirus (Ad), a control adenovirus, or a DDB1 expression virus (b). Fifteen hours after infection, the cells were subjected to UV irradiation (12 J/m2). Three hours postirradiation, the cells were treated with cycloheximide (chx) for the indicated time periods. Extracts (100 μg) of the cells were analyzed for the levels of p53S18P by Western blot experiments.

FIG. 8.

Increased expression of p21Cip1 in DDB2−/− MEFs or DDB2-deficient HeLa cells following UV irradiation. Wild-type or DDB2−/− MEFs (a) were subjected to UV irradiation (12 J/m2) and then maintained in 10% fetal bovine serum medium for the indicated time periods. (Right) Total RNA was analyzed by quantitative real-time PCR for the levels of p21Waf1/Cip1 mRNA as described in Materials and Methods. Extracts (50 μg) were subjected to Western blot assays. The blots were probed with antibodies against p21Cip1 and Cdk2 (left). (b) HeLa cells were transfected with empty vector (control) or vector expressing DDB2 shRNA to isolate stable clones. Cells were treated with UV irradiation at 12 J/m2. Three hours following UV irradiation, cycloheximide (chx) was added to block new protein synthesis. At the indicated time points, cells were harvested and extracts were assayed for p53S18P (left). (Right) Total RNA from the HeLa cells after UV irradiation was subjected to quantitative real-time PCR to assay the levels of p21Waf1/Cip1 mRNA. (c) HeLa cells expressing DDB2 shRNA were transfected with control (Cont) siRNA or p53 siRNA. The transfected cells were treated with UV irradiation. Three hours following UV treatment, cells were harvested and total RNAs were subjected to quantitative real-time PCR for the levels of p21Waf1/Cip1 mRNA.

FIG. 9.

Repair deficiency in DDB2−/− MEFs is reversed by partial depletion of p21Waf1/Cip1. DDB2−/− MEFs were infected with lentivirus expressing shRNA against p21Waf1/Cip1 or a control shRNA. Three days following infection, part of the infected cells were subjected to a Western blot assay with an antibody against p21Waf1/Cip1 or tubulin (loading control). Relative levels of p21Cip1 were quantified, and the results are shown in panel a. The rest of the infected cells were subjected to UDS assays, and the numbers of grains per nucleus from 15 nuclei are plotted in panel b.

FIG. 10.

Deletion of the gene for p21Waf1/Cip1 reverses the repair deficiency in DDB2−/− MEFs. MEFs from wild-type (WT), DDB2−/−, p21−/−, and DDB2−/− p21−/− embryos were subjected to UDS analyses as described in Materials and Methods. After quantification, the numbers of grains per nucleus were plotted. Representative nuclei are shown.

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The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1 - PubMed (original) (raw)