Hum

Hum. lesions, e.g. UV-induced photolesions including cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP), from the genome of UV exposed cells (1). NER includes two distinct subpathways, global genomic repair (GGR) which removes lesions from the entire genome, whereas transcription coupled repair (TCR) eliminates TCN 201 the DNA damage in the transcribed strand of actively transcribing genes (2). An autosomal recessive disorder, Xeroderma Pigmentosum (XP) exhibits impaired NER activity. XP patients are classified into seven groups (XP-A to -G), and the defects of corresponding seven genes (to strain DH5 transformed with either pGEX4T-1 or pGEX-hSug1 (kindly provided by Dr Andrew Paterson, The University of Alabama at Birmingham, Birminghan, AL, USA). After purification, GST and GST-hSug1 were separately incubated with glutathione Sepharose 4B beads (Amersham Bioscience, Uppsala, Sweden) at 4C for 2?h in PBS. The nuclear extract from OSU-2 cells were prepared by incubating OSU-2 cells in nuclear extract (NE) buffer (20?mM HEPES, pH 7.9, 25% glycerol, 0.42?M NaCl, 1.5?mM MgCl2, 0.2?mM EDTA, protease inhibitor cocktail) for 20?min and NE was collected by centrifugation. GST or GST-hSug1 bound beads were incubated with either NE, or purified recombinant XPC (a gift of Dr Yue Zou, East Tennessee State University, Johnson City, TN, USA) at 4C for 2?h in NE buffer. After washing five times with NE buffer, the beads were boiled in 2SDS loading buffer for 5?min and the supernatant was subjected to western blot analysis. siRNA transfection Cul4A and DDB1 siRNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO, USA) in a purified and annealed duplex form. The sequences targeting Cul4A and DDB1 were 5-GAACAGCGAUCGUAAUCAAUU-3 and 5-UAACAUGAGAACUCUUGUC-3, respectively. Specific TCN 201 and control siRNA transfections were performed with Lipofectamine 2000 (Invitrogen) according to manufacture’s instruction. Immuno-slot blot analysis The amount of CPD in DNA was quantified with non-competitive immuno-slot blot assay. Briefly, XP-C cells in 100?mm plates were transiently co-transfected with DDB2 and either empty vector, wild type or K655A XPC mutant. Twenty-four hours post-transfection, cells were split into 60?mm plates and grown for an additional 24?h. After UV exposure (10?J/m2) and desired incubation periods, cells were recovered by trypsinization and immediately lysed for DNA isolation. The identical amounts of DNA samples were loaded on nitrocellulose membranes and the amount of CPD was detected with monoclonal anti-CPD antibody (TDM-2). The intensity of each band was determined by laser densitometric scanning and the amount of damage remaining, compared with the initially induced DNA damage, was used to calculate the relative repair rates. RESULTS XPC is degraded following UV irradiation Our previous studies have indicated Mouse monoclonal to CD47.DC46 reacts with CD47 ( gp42 ), a 45-55 kDa molecule, expressed on broad tissue and cells including hemopoietic cells, epithelial, endothelial cells and other tissue cells. CD47 antigen function on adhesion molecule and thrombospondin receptor that the level of XPC in cells decreases upon UV irradiation (27). To further confirm this phenomenon, we compared the decay rates of XPC in UV- or mock-irradiated normal human fibroblast, OSU-2 cells pre-treated with cycloheximide (CHX), an inhibitor of protein synthesis. Figure 1A shows that in the absence of new XPC synthesis, XPC protein exhibits a high decay rate following UV irradiation, and the distinct pattern of XPC degradation could be observed TCN 201 as early as 30?min after UV treatment (lanes 5C8). To rule out the possibility that the decreased XPC level at 125?kDa is due to the conversion of XPC to slower migrating modified forms, we over exposed the film to show the corresponding levels of various XPC bands. As demonstrated TCN 201 in Number 1A, the changes of XPC is not fully obvious at 2? h time point and yet the level of XPC at 125?kDa is lower than that seen at 1?h time.