Abstract
Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.
Highlights
UV light-induced cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6 – 4) photoproducts as well as chemical carcinogen-induced lesions [1]
The Effects of C-terminal Deletion of CS group B protein (CSB) on the Function in transcription-coupled nucleotide excision repair (TC-NER)—Because CSB interacts with polymerase II (Pol II) in a UV light-dependent manner (14 –16), we examined whether the mutant CSB proteins interact with Pol II after UV irradiation
When UV light-induced lesions occur at the transcribed strands of actively transcribed genes, Pol II is arrested at the damaged sites [37, 38], and TC-NER is initiated
Summary
Expression Constructs and Stable Cell Lines—To generate epitope-tagged CSB expression constructs, WT and mutant CSB cDNA fragments were amplified by PCR and cut with XhoI at the 5Ј end and with XbaI at the 3Ј end. Preparation of Whole Cell Extracts—Cells (1.0 ϫ 106) were washed once with PBS and lysed with 100 l of SDS-PAGE sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromphenol blue, and 2.5% mercaptoethanol) by boiling for 5 min. Interaction of CSB with Pol II—After irradiation with 20 J/m2 UV light, cells were incubated for 30 min and harvested. CS1ANSV cells expressing CSB were irradiated with 20 J/m2 UV light, incubated for 1 h, and treated with CSK-Triton buffer to prepare the insoluble fractions (CSK-ppt fraction). After washing three times with CSK-Triton buffer, 1ϫ SDS-PAGE sample buffer was added to the pellet and boiled for 5 min. Cells were irradiated with UV light 24 – 48 h after forward transfection, incubated for the indicated amount of time, and harvested. A monoclonal anti-HA tag (catalog no. 11867423001) antibody was from Roche
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