Abstract
UV-sensitive syndrome is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair that rapidly removes transcription-blocking DNA damage. UV-sensitive syndrome consists of three genetic complementation groups caused by mutations in the CSA, CSB, and UVSSA genes. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, which is required for stabilization of Cockayne syndrome group B (CSB) protein and reappearance of the hypophosphorylated form of RNA polymerase II after UV irradiation, forms a complex with ubiquitin-specific peptidase 7 (USP7). In this study, we demonstrated that the deubiquitination activity of USP7 is suppressed by its interaction with UVSSA. The interaction required the tumor necrosis factor receptor-associated factor domain of USP7 and the central region of UVSSA and was disrupted by an amino acid substitution in the tumor necrosis factor receptor-associated factor-binding motif of UVSSA. Cells expressing mutant UVSSA were highly sensitive to UV irradiation and defective in recovery of RNA synthesis after UV irradiation. These results indicate that the interaction between UVSSA and USP7 is important for TC-NER. Furthermore, the mutant UVSSA was rapidly degraded by the proteasome, and CSB was also degraded after UV irradiation as observed in UVSSA-deficient cells. Thus, stabilization of UVSSA by interaction with USP7 is essential for TC-NER.
Highlights
Bulky, helix-distorting DNA lesions, including UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone [] photoproducts (6-4PPs) [1]
We examined the effect of interaction with UV-stimulated scaffold protein A (UVSSA) on the deubiquitination activity of ubiquitin-specific peptidase 7 (USP7) and identified the regions in both proteins required for binding
USP7(1–564) and USP7(1–208) as well as full-length USP7 (USP7(1–1102)) co-precipitated with UVSSA, whereas USP7(208 –1102) did not (Fig. 3B). These results indicate that UVSSA interacts with the N-terminal region of USP7 containing the tumor necrosis factor receptor-associated factor (TRAF) domain
Summary
Generation of Plasmids and Baculoviruses for Protein Expression—For expression of WT and truncated UVSSA, DNA sequence encoding FLAG and HA tags was attached to the 5Ј-end of each UVSSA cDNA, and the resultant fragments were inserted between the NotI and XbaI sites of vector pFastBac-1 (Invitrogen). Cells infected with recombinant baculovirus for FLAG-HA-UVSSA or co-infected with viruses for FLAG-HA-UVSSA and His6-V5-USP7 were lysed in NETN buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM DTT, and Complete protease inhibitor mixture (Roche Applied Science)) at 4 °C for 30 min. The cell lysates were clarified by centrifugation at 17,600 ϫ g for 10 min and incubated with protein G-Sepharose (GE Healthcare) at 4 °C for 1 h. Cells infected with recombinant baculovirus for His6V5-USP7 were lysed in TALON buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5% Triton-X-100, 10% glycerol, and Complete protease inhibitor mixture) at 4 °C for 30 min. The cell lysates were clarified by centrifugation at 17,600 ϫ g for 10 min, and the resultant supernatant was incubated with protein G-Sepharose at 4 °C for 1 h.
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