DCAF7 is required for maintaining the cellular levels of ERCC1-XPF and nucleotide excision repair

Abstract

The ERCC1-XPF heterodimer is a structure-specific endonuclease and plays multiple roles in various DNA repair pathways including nucleotide excision repair and also telomere maintenance. The dimer formation, which is mediated by their C-terminal helix-hairpin-helix regions, is essential for their endonuclease activity as well as the stability of each protein. However, the detailed mechanism of how a cellular level of ERCC1-XPF is regulated still remains elusive. Here, we report the identification of DDB1- and CUL4-associated factor 7 (DCAF7, also known as WDR68/HAN11) as a novel interacting protein of ERCC1-XPF by mass spectrometry after tandem purification. Immunoprecipitation experiments confirmed their interaction and suggested dominant association of DCAF7 with XPF but not ERCC1. Interestingly, siRNA-mediated knockdown of DCAF7, but not DDB1, attenuated the cellular level of ERCC1-XPF, which is partly dependent on proteasome. The depletion of TCP1α, one of components of the molecular chaperon TRiC/CCT known to interact with DCAF7 and promote its folding, also reduced ERCC1-XPF level. Finally, we show that the depletion of DCAF7 causes inefficient repair of UV-induced (6-4) photoproducts, which can be rescued by ectopic overexpression of XPF or ERCC1-XPF. Altogether, our results strongly suggest that DCAF7 is a novel regulator of ERCC1-XPF protein level and cellular nucleotide excision repair activity.

Keywords: DCAF7/WDR68/HAN11; ERCC1-XPF; Nucleotide excision repair; TRiC/CCT.

Figure 1.. DCAF7 interacts with XPF but not ERCC1

(A and B) DCAF7-Myc and/or FLAG-ERCC1 were ectopically expressed in XP2YO(SV) cells and cell lysates were immunoprecipitated by anti-FLAG antibody resin (A) or anti-Myc antibody resin (B). (C and D) FLAG-DCAF7 and/or Myc-tagged XPF (C) or non-tagged XPF (D) were expressed in XP2YO(SV) cells and precipitated by either anti-Myc (C) or anti-FLAG (D) antibody resin. (E) The same amounts of Non-tagged ERCC1 and FLAG-DCAF7 expression plasmids (0.65 μg each) were co-transfected with an increasing amount (0.65–5.2 μg) of XPF-Myc expression plasmid into XP2YO(SV) cells. The total amount of DNA was adjusted with empty vector pEF6/Myc-His. After 48 h, cell lysates were prepared and immunoprecipitated by anti-FLAG antibody resin. Each sample was analyzed by western blotting with the indicated antibodies.

Figure 2.. Depletion of DCAF7 or TCP1α reduces the cellular level of ERCC1-XPF.

(A) U2OS cells were transfected with DCAF7 siRNA (#1) and incubated for the indicated period. After cell lysis, each sample was analyzed by western blotting with the indicated antibodies (left panel). The signal intensity of each band was quantified and adjusted using internal control β-actin (right panel). (B - E) U2OS cells were transfected with either control siRNA or DCAF7 siRNA (#1) (B) or TCP1α siRNA (#1) (D). After 72 h, cell lysates were prepared and analyzed by western blotting with the indicated antibodies. An asterisk indicates non-specific bands. The impacts of DCAF7 or TCP1α knockdown on ERCC1-XPF level were tested three times, and the mean and SD were shown in (C) or (E), respectively.

Figure 3.. The downregulation of ERCC1-XPF level by DCAF7 depletion is independent of Cul4-DDB1 E3 ligase, but partly dependent on 26S proteasome.

(A and B) U2OS cells were transfected with DDB1 siRNA (A) or various combinations of control siRNA, DDB1 siRNA and DCAF7 siRNA (#1) (B) and incubated for 72 h. (C and D) U2OS cells were transfected with DCAF7 siRNA (#1) and incubated for 12 h (C) or 60 h (D). The cells were treated with MG-132 (final 10 μM) or DMSO for 12 h and lysed for western blotting with the indicated antibodies. The relative band intensities adjusted using internal control β-actin were shown below each band.

Figure 4.. Depletion of DCAF7 suppresses cellular NER activity.

(A) HeLa cells were transfected with DCAF7 siRNA (#1) and incubated for 72 h. The cells were irradiated with 10 J/m² of UV-C and further incubated for 1 or 2 h. Genomic DNA was purified from each sample and analyzed for the repair kinetics of 6–4PP using ELISA. Each point represents the mean of three experiments and bars indicate the SD (left panel). Knockdown of DCAF7 and subsequent downregulation of ERCC1-XPF were also monitored by western blotting of cell lysates prepared from the same samples (right panel). The relative band intensities adjusted using internal control β-actin were shown below each band. (B) Flp-In T-REx 293/FLAG-XPF-6xHis cells were transfected with DCAF7 siRNA and incubated for 48 h. Doxycycline was added to the cell culture at a final concentration of 1 μg/ml and cells were further incubated for 24 h. After irradiation with 10 J/m² of UV-C, the repair kinetics of 6–4PP (left panel) and the protein levels of DCAF7, XPF and ERCC1 (right panel) were determined as described in (A).