Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Abstract

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.

Keywords: CRL4; Cdt2; PCNA; XPG; gap-filling DNA synthesis; nucleotide excision repair; protein degradation; ubiquitylation.

Figure 1.

(See previous page). XPG is degraded upon DNA damaging agent treatment. (A and B) UV and cisplatin treatment causes a reduction of XPG protein level. HeLa cells were treated with UV (A) or cisplatin (B) for the indicated time periods. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression of XPG. Tubulin was also detected as a loading control. (C) XPG protein exhibits a high decay rate following UV irradiation in the absence of new XPG synthesis. HeLa cells were pretreated with CHX for 0.5 h, UV irradiated and further cultured in the presence of CHX for various time periods. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG. Tubulin was also detected as a loading control. (D) XPG is degraded by proteasome upon UV irradiation. HeLa cells were pretreated with MG132 for 0.5 h, UV irradiated and further cultured in the presence of MG132 for various time periods. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG. Lamin B was used to serve as a loading control. The levels of total XPG in each lane were quantified and normalized to the loading control and then to the initial amount of XPG. Data from 3 independent experiments were plotted on the right of each figure.

Figure 2.

UV-induced XPG degradation is dependent on efficient NER. NER-proficient human fibroblast OSU-2 cells (A) and various NER-deficient human fibroblasts (B–F) were UV irradiated at 10 J/m² and further cultured for the indicated time periods. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG. Tubulin was used to serve as a loading control. XPG bands were scanned, quantified, normalized to the tubulin and then to the initial amount of XPG. Data from 3 independent experiments were plotted on the bottom of reach figure.

Figure 3.

(See previous page). DDB2 indirectly participates in XPG degradation upon DNA damage. (A–C) DDB2 is required for UV-induced, but not cisplatin-induced XPG degradation. DDB2-deficient 041 cells and DDB2 stably transfected 041-N22 cells were UV irradiated at 10 J/m², further cultured for the indicated time periods. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG. Actin was used to serve as a loading control. (A) HeLa cells were transfected with either control or 2 different DDB2 siRNA for 48 h, UV irradiated at 20 J/m² (B) or treated with cisplatin at 100 μM for 1 h. (C) Cells were further cultured for 2 h, whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG and DDB2. Tubulin was used to serve as a loading control. (D) Both DDB1 and Cul4A are required for cisplatin-induced XPG degradation. HeLa cells were transfected with either control siRNA or siRNA specific to DDB1, or Cul4A for 48 h, then treated with cisplatin at 100 μM for 1 h. Cells were further cultured for 2 h, whole cell lysates were prepared and subjected to immunoblotting to detect the expression level of XPG, DDB1, and Cul4A. Tubulin was used to serve as a loading control. The levels of total XPG in each lane were quantified and normalized to the loading control and then to the initial amount of XPG. Data from 3 independent experiments were plotted on the right.

Figure 4.

Cdt2 is responsible for XPG degradation upon DNA damage. (A and B) Cdt2 is recruited to the UV-induced DNA damage sites. HeLa cells were UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium for the indicated time periods. The cells were fixed and double immunostained with anti-XPG (red) and anti-Cdt2 (green) antibodies (A), as described in Materials and Methods. The total number of cells with foci was counted from at least 5 separate fields, and the percentage of cells with foci was calculated and plotted. (B) Bar: SD. (C–E) Cdt2 is required for UV and cisplatin-induced XPG degradation. HeLa cells were transfected with either control or Cdt2 siRNA for 48 h, UV irradiated at 40 J/m² or treated with cisplatin at 100 μM for 1 h. Cells were further cultured for 2 h, whole cell lysates were prepared and subjected to immunoblotting to detect the expression levels of XPG and Cdt2. Tubulin was used to serve as a loading control. The levels of total XPG in each lane were quantified and normalized to the tubulin and then to the initial amount of XPG. Data from 3 independent experiments were plotted at the bottom. (C) HeLa cells growing on coverslips were transfected with either control or Cdt2 siRNA for 48 h, UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium for 0.5, 2, 4 and 8 h. The cells were fixed and double immunostained with anti-CPD (green) and anti-XPG (red) antibodies. (D) The total numbers of XPG and CPD foci were counted from at least 5 separate fields, the ratios of XPG to CPD foci were calculated and plotted. (E) Bar: SD, *: P < 0.05 compared with siCtrl 2 h.

Figure 5.

PCNA is required for Cdt2-mediated XPG degradation by mediating the interaction between XPG and Cdt2 upon UV irradiation. (A) PCNA is required for UV-induced XPG degradation. HeLa cells were transfected with either control or 2 different PCNA siRNA for 48 h, UV irradiated at 20 J/m². Cells were further cultured for 2 h. Whole cell lysates were prepared and subjected to immunoblotting to detect the expression levels of XPG and PCNA. Tubulin was used to serve as a loading control. The levels of total XPG in each lane were quantified and normalized to the loading control and then to the initial amount of XPG. Data from 3 independent experiments were plotted at the bottom. (B) PCNA is required for the interaction between XPG and Cdt2 upon UV irradiation. HeLa cells growing on coverslips were transfected with either siCtrl or siPCNA-1 for 48 h, UV irradiated at 20 J/m², and further cultured for 2 h. Cells were fixed and subjected to in situ PLA analysis. Primary mouse anti-XPG and rabbit anti-Cdt2 antibodies were combined with secondary PLA probes as described in the Materials and Methods. The interaction events are visible as red dots (nuclear staining in blue).

Figure 6.

(See previous page). Cdt2-mediated XPG degradation facilitates the recruitment of DNA Pol δ to UV-damaged sites and subsequent gap-filling DNA synthesis. (A and B) Cdt2 is not required for the removal of UV-induced DNA lesions. HeLa cells were transfected with either control siRNA or siCdt2 for 48 h. Cells were UV irradiated at 10 J/m², and further cultured for the indicated time periods. Genomic DNA was isolated and the same amount of DNA was loaded for ISB with anti-CPD or anti-6-4PP antibody. Single strand DNA (ssDNA) was detected to serve as a loading control. (A) The intensity of each band was scanned and plotted. (B) (C and D) Cdt2 is required for the gap-filling DNA synthesis upon UV irradiation. HeLa cells growing on coverslips were transfected with either control or Cdt2 siRNA for 48 h, UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium containing BrdU for 2 and 4 h. The cells were fixed and double immunostained with anti-CPD (green) and anti-BrdU (red) antibodies. (C) The total numbers of CPD and BrdU foci in non-S phase cells were counted from at least 5 separate fields, the ratios of BrdU to CPD foci were calculated and plotted. (D) Yellow arrow: S phase cells. Bar: SD, *: P < 0.01 compared with their corresponding siCtrl. (E and F) Cdt2 is required for the recruitment of DNA Pol δ to UV damaged sites. siCtrl and siCdt2 transfected HeLa cells were UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium for 1 h. The cells were fixed and double immunostained with anti-XPB (green) and anti-p125 (red) antibodies. (E) The total numbers of XPB and p125 foci were counted and the ratios of p125 to XPB foci were calculated and plotted. (F) n = 5, Bar: SD, *: P < 0.01 compared with siCtrl.