PMC

ATR is the major kinase responsible for cellular XPA phosphorylation after UV irradiation. A, A549 cells were mock treated (lane 1) or treated with 20 J/m² UV irradiation, and then further incubated for 4 hours in the presence of 100 Amol/L wortmannin (Wort; lane 4) or 10 mmol/L caffeine (Caff; lane 3) before harvesting. Total cell lysates were used for Western blot analysis with anti-XPA, anti-ATR, and anti-ATM, respectively. B, A549 cells were transfected with ATR, ATM, or green fluorescent protein (GFP) siRNA as described in Materials and Methods. Total cell lysates were harvested 72 hours after transfection and probed with the indicated antibodies, respectively. C, A549 or HeLa cells were transfected with indicated siRNA and then treated with 20 J/m² UV irradiation at 72 hours after transfection. Total cell lysates were immunoblotted with anti-XPA and antiactin antibodies, respectively. D, ATR- and ATM-deficient cells were treated with the indicated doses of UV and total cell lysates were prepared at 4 hours after treatment for Western blotting with anti-XPA and antiactin, respectively.

Identification of XPA phosphorylation sites. A, XPA-deficient cells were transfected with expression constructs of XPA-WT, XPA-S173A, XPA-S196A, and XPA-S173.196A, respectively. The stably transfected cells were selected as described in Materials and Methods. Total cell lysates were prepared and incubated with Nickel-NTA resin (Qiagen) for 1 hour at 4°c. After centrifugation, the precipitates were washed thrice with PBS containing 0.05% NP40 and once with kinase buffer. The precipitates were resuspended in 20 AL kinase buffer and the kinase reactions were done for 30 minutes at 30°c in the presence of 100 units DNA-PK and [γ-³²P]ATP. The radiolabeled proteins were visualized following SDS-PAGE (top). The precipitated His-XPA was confirmed by Western blotting with anti-XPA antibody (bottom). B, transfected XPA cells were grown overnight in phosphate-depleted medium before irradiation with 20 J/m² UV. Then, ³²P-labeled orthophosphoric acid was added and cells were further incubated for 8 hours before harvest. Total cell lysates were prepared and precipitated with Nickel-NTA resin at 4°c. After extensive washing, the precipitates were separated on SDS-PAGE and radiolabeled proteins were detected (top). Precipitated exogenous XPA was probed by Western blotting (bottom). C, XPA-deficient cells were stably transfected with XPA-WT, XPA-S173D, and XPA-S196D constructs, respectively. Total cell lysates were prepared and probed with anti-XPA and antiactin antibodies, respectively.

Phosphorylation of XPA in vitro. A, equal amount (40 pmol) of RPA (lane 1) and XPA (lanes 2-4) were incubated in a 20-μL reaction, respectively, with 50 units (lanes 1 and 3) or 100 units (lane 4) of DNA-PK for 30 minutes at 30°C in the presence of [γ³²P]ATP. The radiolabeled proteins were visualized using PhosphorImager following SDS-PAGE. B, the kinase assays were done in 20-μL reaction system containing 200 nmol/L XPA, 100 Amol/L cold ATP, and 50 to 200 units of DNA-PK for 30 minutes at 30°c. The phosphorylated XPA was visualized by Western blotting (W.B.) with anti-XPA antibody. C, the immunoprecipitation kinase assays were done as described in Materials and Methods. Cells were first treated with 40 J/m² UV irradiation, and then endogenous ATR and ATM were immunoprecipitated by anti-ATR and ATM antibodies, respectively. The ATR and ATM immunoprecipitates were then resuspended in 20 μL of kinase buffer containing 10 μCi of [γ-³²P]ATP and 1 μg of purified His-XPA protein and incubated at 30°C for 10 to 30 minutes. The radiolabeled XPA was visualized following SDS-PAGE (top). Immunoprecipitated endogenous ATM or ATR was confirmed by Western blotting (bottom). D, the kinase assays were done as above except that the XPA peptides, XPA173 and XPA196, were used. The control peptide was the XPA173 peptide with a substitution of the serine to alanine.

UV and camptothecin (CPT) sensitivities of cells with XPA mutants. A, transfected XPA cells were seeded in 96-well plates and allowed to attach overnight. The cells were then treated with indicated doses of UV irradiation. After treatment, cells were washed once with PBS and further incubated for 72 hours. Aliquots of 10 μL of MTT (5 mg/mL) were then added to each well. After 4 hours of incubation, intensity of the formed color was quantitated by a spectrophotometric plate reader at 490 nm after solubilization in 150 AL of DMSO. Data were normalized to the controls (as the value of 1). Points, mean of four different measurements; bars, SD. B, cells were incubated with indicated doses of camptothecin for 8 hours, and then washed with PBS and further cultured for 24 hours. The MTT assays were done as above. C, colony formation assays of cells with phosphorylation-deficient XPA mutants following UV irradiation. XPA-deficient cells were transfected with constructs for cellular expression of XPA-WT, XPA-S173A, XPA-S196A, and XPA-S173.196A, respectively. The stably transfected cells were obtained as described in Materials and Methods. Five hundred cells were then plated to each dish. After overnight attachment, cells were irradiated with indicated doses of UV and then allowed to grow for 2 weeks. The colonies formed were counted under microscope. The data were normalized to the controls (as the value of 1). Points, mean of three different measurements; bars, SD. D, colony formation assays were done as described in (C).

XPA is phosphorylated in cells on UV irradiation. A, A549 cells were treated with 20 J/m² UV or mock treated. Total cell lysates were harvested at 4 hours after UV irradiation and then treated with 200 units of CIAP (Promega) for 1 hour at 37°c in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of 50 mmol/L glycerophosphate (G.P.) or mock treated (lanes 1 and 2). The treated cell lysates were then subjected to Western blotting and probed with anti-XPA (a) and anti-RPA32 (b), respectively. c, cells were treated with 20 J/m² UV or mock treated and total cell lysates were harvested for immunoprecipitation assays with anti-XPA antibody. The immunoprecipitated XPA was treated with CIAP or mock treated and then analyzed by Western blotting with anti-XPA antibody. B, A549 cells were grown overnight in phosphate-depleted medium before irradiation with 20 J/m² UV. Then, ³²P-labeled orthophosphoric acid was added and cells were further incubated for 8 hours before harvest. Immunoprecipitation assay was done with anti-XPA antibody. Immunoprecipitates were separated on SDS-PAGE and radiolabeled proteins were detected (left). Immunoprecipitated endogenous XPA was probed by Western blotting (right). C, cells were UV irradiated or mock treated and then cytoplasmic and nuclear extractions were separated on SDS-PAGE for Western blot analysis using anti-XPA antibody. D, a, cells were irradiated with 20 J/m² of UV and the cytoplasmic fraction (lane 1) was isolated. The nuclear pellet was then sequentially extracted with buffer of increasing salt concentration (lanes 2-5). NM, nuclear matrix (lane 6). b, nuclear extracts were prepared at indicated times after 20 J/m² UV treatment of cells. c, cells were irradiated with indicated doses of UV and the nuclear extracts were prepared at 4 hours after UV treatment.

ATR interaction and colocalization with XPA in cells after UV irradiation. A, total cell lysates prepared from UV-treated or mock-treated A549 cells were used for coimmunoprecipitation assays with anti-ATR antibody. Proteins from the immunoprecipitates were detected by Western blotting using anti-XPA and anti-ATR antibodies. As controls, 10% of the total volumes of the whole cellular lysates used for the coimmunoprecipitation were also included. B, top, total cell lysates prepared from A549 cells were used for coimmunoprecipitation assays with anti-XPA antibody; bottom, whole-cell extracts prepared from 2 × 10⁶ cells were mixed with 2 μg of His-XPA and incubated at 4°c for 10 to 14 hours. The XPA-bound proteins were probed by anti-ATR antibody. C, total cellular lysates were incubated with anti-ATR antibodies for 4 to 6 hours, followed by 1-hour incubation with protein A/G-agarose beads. The immunoprecipitates were washed thrice with PBS containing 0.5% NP40 and further incubated with the buffer of high concentration of salt [15 mmol/L Tris-Cl (pH 7.5), 0.6 mol/L NaCl, 0.1% NP40] for 30 minutes at 4°c. After centrifugation and washing, purified His-XPA was added and further incubated in 500 AL of XPA binding buffer [40 mmol/L HEPES-KOH (pH 7.5), 75 mmol/L KCl, 8 mmol/L MgCl₂, 1 mmol/L DTT, 5% glycerol and 100 Ag/mL bovine serum albumin, 0.1% NP40] for 4 to 6 hours. The bound proteins were detected by Western blotting with anti-XPA antibody. LC, loading control (20 ng purified His-XPA). D, cells were treated with 20 J/m² UV or mock treated, followed by 4-hour incubation. After extraction of cytoplasmic proteins with PBS containing 0.5% NP40, cells were fixed and incubated with anti-XPA and anti-ATR antibodies. Cells were then stained with fluorescent dye-linked secondary antibodies and visualized by fluorescence microscopy. b and f, red, anti-ATR stained cells; c and g, green, anti-XPA stained cells; d and h, merged images of the anti-XPA and anti-ATR stained cells; a and e, 4‘,6-diamidino-2-phenylindole-stained nuclei.