Cockayne syndrome group A and N (CSB) protein act in transcription-coupled restoration, a subpathway of nucleotide excision restoration. proteasomal RPN2, and Sug1 in the chromatin area. Remarkably, both biochemical inhibition and hereditary defect of VCP/p97 enhance the recovery of RNA synthesis following UVR, whereas both VCP/p97 and proteasome inhibitions decrease cell viability. Our findings reveal a new role of VCP/p97 segregase in the timely processing of ubiquitinated CSB from damaged chromatin. because of VCP/p97 inhibition, leads to retention of CSB, UBXD7, and proteasomal proteins Nebivolol HCl manufacture in chromatin. Compromising VCP/p97 function does not impair post-TCR RRS but severely Siglec1 affects cell viability upon UV exposure. Thus, our findings reveal a key role of VCP/p97 in processing ubiquitinated CSB during TCR and in the maintenance of cell viability by clearance of unstable subunit(s), including CSB, from macromolecular protein complexes on damaged chromatin. Experimental Procedures Chemicals, Antibodies, and Cell Lines Nebivolol HCl manufacture The VCP/p97 inhibitor N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), was obtained from Sigma-Aldrich (St. Louis, MO). The proteasome inhibitor MG132 was purchased from EMB Millipore (Billerica, MA). Anti-FLAG M2 and anti-Myc-agarose affinity gels were purchased from Sigma-Aldrich. Anti-VCP/p97, anti-UFD1, anti-NPL4, anti-H2AX, anti-ubiquitin FK2, anti-Myc, and the antibodies against RPN2/S1 and Sug1 were purchased from Abcam, BD Biosciences, Abnova/Novus Biologicals (Littleton, CO), Cell Signaling Technology (Danvers, MA), EMB Millipore, Santa Cruz Biotechnology (Dallas, TX) and Thermo Scientific (Rockford, IL), Nebivolol HCl manufacture respectively. HCT116 and HCT116-USP7?/? cells were obtained from the laboratory of Bert Vogelstein (33). U2OS cell lines, stably transfected with tetracycline-inducible DNA constructs expressing Myc-tagged WT or mutant EQ (E578Q)-VCP/p97, were provided by the Weihl laboratory (34). The dominant-negative EQ mutant is deficient in ATP hydrolysis activity of VCP/p97 but still able to bind ubiquitinated substrates. The CSB-defective CS1AN cell line, which expresses CSB under control of the tetracycline-responsive promoter (CS1AN+iCSB cells) (35), was a gift from Dr. Yonggang Zhou. HeLa cells stably expressing C-terminally FLAG-HA-tagged CSA (HeLa CSA.Com cells) (36) were provided by the Nakatani laboratory. The VCP/p97 mutant fibroblasts GM22764 and GM22041 from clinically affected donor subjects heterozygous for mutated VCP/p97 genes were obtained through the Coriell Cell Repository (Camden, NJ). GM22764 fibroblasts harbor a 475C>T transition in exon 5 of the VCP gene, resulting in the substitution of cysteine for arginine at codon 159 (R159C), whereas GM22041 fibroblasts harbor a 463C>T transition in exon 5 of the VCP/p97 gene, resulting in the substitution of cysteine for arginine at codon 155 (R155C). These VCP/p97 mutations cause inclusion body myopathy with early-onset Paget disease and frontotemporal dementia. RNA Interference The siRNA sequences for non-coding 5-AAUUCUCCGA-ACGUGUCACGU-3 (catalog no. SI03650325);UBXD1, 5-CGCCTCCATCATGAAGATCTA-3 (catalog no. SI04209044); UBXD7, 5-CAGC-ACGTGCATATTCATTTA-3 (catalog no. SI00455364); UFD1, 5-CACTGGATGATGC-AGAACTTA-3 (catalog no. SI04132583); and VCP/p97, 5-AACAGCCAUUCUCAAACAG-AA-3 (catalog no.SI03019730) were purchased from Qiagen (Valencia, CA). The siRNA for CSB/ERCC6, 5-GTGTGCATGTGTCTTA-CGA-3 (Am16708), was obtained from Life Technology. Cell Culture, DNA, and siRNA Transfection HCT116 and HCT116-USP7?/? cells were grown in McCoy’s 5A medium. CSB-defective CS1AN cell lines, U2OS, HeLa cells, and their derivatives were grown in DMEM. Human fibroblasts were grown in minimum Eagle’s medium with additional amino acids. All cells were grown in medium supplemented with 10% (or 15% if required) FBS, penicillin, and streptomycin at 37 C in a humidified atmosphere of 5% CO2. Hygromycin B and zeocin were used for maintaining stably transfected CSB-defective CS1AN cell lines and Myc-tagged VCP/p97 U2OS cell lines. Plasmid DNAs were transfected into U2OS or CS1AN cell lines using FuGENE 6 transfection reagents (Promega, Madison, WI). The siRNA transfection was conducted using Lipofectamine 2000 reagent (Life Technologies) as described earlier (37, 38). Cellular Protein Fractionation The experiment was conducted as described by Anindya R. (24) with modifications. Briefly, cells (107) were lysed with Nebivolol HCl manufacture 1 ml (5 cell volume) of cytoplasmic lysis buffer (10 mm Tris-HCl (pH 7.9), 0.34 m sucrose, 3 mm CaCl2, 2 mm magnesium acetate, 0.1 mm EDTA, 1 mm DDT, 0.5% Nonidet P-40, and a protease inhibitor mixture). Nuclei were pelleted by centrifugation at 3500 for 15 min and washed with cytoplasmic lysis buffer Nebivolol HCl manufacture without Nonidet P-40 and then lysed in 1 ml of nuclear lysis buffer (20 mm HEPES (pH 7.9), 3 mm EDTA, 10% glycerol, 1.5 mm MgCl2, 150 mm KOAc, and protease inhibitors). The nucleoplasmic fractions were separated by centrifugation at 15,000 for 30 min, and the pellets were designated as the chromatin fraction. For further processing, the pellets were resuspended in 0.2 ml of nuclease incubation buffer (150 mm HEPES (pH 7.9), 1.5 mm MgCl2, 150 mm KOAc, and protease inhibitors) and incubated with 50 units Benzonase (25 units/l) for 30 min at room temperature. The soluble chromatin fraction was collected by centrifugation at 20,000 for 30 min, whereas the insoluble chromatin fraction was dissolved by boiling in SDS sample.