DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

Abstract

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin-proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein.

Figure 1

Multiple high-molecular-weight bands of XPC resulted from UV irradiation of normal human cells. OSU-2 cells were UV irradiated at 0, 5, 10, 20 and 50 J/m² and incubated for 1 h (A) or irradiated at 20 J/m² and incubated for indicated times (B). Cell lysates were prepared by boiling in SDS lysis buffer as described in the Materials and Methods. Total protein (50 μg) was loaded for SDS–PAGE and detected by western blotting with rabbit anti-XPC antibody (XPC-2, 1:5000).

Figure 2

UV-induced high-molecular-weight bands of XPC are SUMO-1 conjugated (A) OSU-2 cells were UV irradiated at 20 J/m² and incubated for 1 h. The cell lysates were prepared by direct lysis in SDS lysis buffer and diluted in RIPA buffer as described in Materials and Methods. Total protein (2 mg) was subjected to IP with anti-SUMO-1, anti-SUMO-2/3 or anti-Ubiquitin antibody. The precipitated proteins were detected by western blotting with anti-XPC antibody. Whole cell lysates (50 μg) were loaded on SDS–PAGE as input. (B) XP-C cells were transfected with either XPC-V5-His plus SUMO-1-FLAG or XPC-V5-His plus Ub-HA, then UV irradiated at 20 J/m² and allowed to repair for 1 h. Total protein (2 mg) was used for pull-down with Ni-NTA agarose beads. The precipitated proteins were detected with anti-XPC, anti-FLAG or anti-HA antibody.

Figure 3

Ubc9 siRNA knocks down the expression of Ubc9 and compromises the UV-induced high-molecular-weight bands of XPC. OSU-2 cells were transiently transfected with either control siRNA or siRNA targeted to Ubc9 for 48 h. The cells were UV irradiated at 20 J/m² and then incubated for another 1 h. The cell lysates were subjected to SDS–PAGE and proteins detected by western blotting with anti-XPC, anti-Ubc9 or anti-Actin antibody. The intensity of each band was determined by densitometry and the relative intensity of each band calculated in reference to control without transfection.

Figure 4

p53 and DDB2 are involved in UV-induced XPC modification by SUMO-1 and ubiquitin (A) OSU-2, p53-deficient 041 and DDB2-deficient XP-E cells were UV irradiated at 20 J/m² and incubated for 1 h. The cell lysates were subjected to western blotting with anti-XPC antibody. (B) and (C) 041 cell lines were transiently transfected with either p53 or DDB2-FLAG construct for 48 h. The cells were irradiated with UV at 20 J/m² and repaired for 1 h. The segregated proteins were detected by western blotting with anti-p53 or anti-DDB2 to confirm the ectopic expression in transfectants (B). The XPC and its modifications were detected with rabbit anti-XPC antibody (C).

Figure 5

UV-induced XPC modifications are compromised in XP-A cells (A) OSU-2, XP-A, XP-F and XP-G cells were UV irradiated at 20 J/m² and incubated for another 1 h. The proteins were subjected to SDS–PAGE and detected with anti-XPC and anti-XPB (loading control) antibodies. (B) XP-A cells were transiently transfected with either empty vector or pcDNA3.1/XPA for 48 h, UV irradiated at 20 J/m² and incubated for another 1 h. The proteins were subjected to SDS–PAGE and detected with anti-XPC, anti-XPA and anti-Actin (loading control) antibodies. (C) H460 cells, expressing either control vector or siRNA targeted to XPA, were UV irradiated at 20 J/m² and incubated for another 1 h. The proteins were subjected to SDS–PAGE and detected with anti-XPC, anti-XPA and anti-XPB antibodies.

Figure 6

Ubiquitin–proteasome system is involved in UV-induced XPC modification. (A) ts20 and its parental cell line A31N were cultured at 32°C or shifted to 39°C for 16 h followed by UV irradiation at 20 J/m² and further incubation for 1 h at 32°C or 39°C. XPC protein and its modifications were detected by immunoblotting with anti-XPC antibody. (B) OSU-2 cells were treated with 10 μM MG132 or mock treated for 1 h and subjected to UV irradiation at 20 J/m². The cell lysates were prepared at indicated time points following UV treatment and analyzed. (C) OSU-2 cells were treated with 10 μM lactacystin or mock treated for 1 h and subjected to UV irradiation at 20 J/m². The cell lysates were prepared after 1 h further incubation and the proteins were detected with anti-XPC antibody.