DNA-agarose was prepared by coupling sheared salmon sperm DNA to CNBr activated Sepharose CL6-B (Pharmacia). The Ku complex was further purified by superdex 200 chromatography. The Ku 70/80 was eluted from the affinity matrix using 10 ml of 1.75 M MgCl 2 in 50% ethylene glycol, 25 mM Tris The DNA-PK CS eluted from the column at 0.2 M KCl and was further purified by gel filtration chromatography using a superdex 200 16/60 column (Pharmacia). Weakly bound proteins were eluted sequentially with 10 ml of a buffer containing 25 mM Tris Briefly, HeLa cell nuclear extract was mixed for 16 h with 2 ml of anti-Ku 80 affinity matrix (2 mg IgG/ml) at 4☌. DNA-PK CS and Ku 70/80 was purified from HeLa cells by immunoaffinity chromatography using an anti-Ku 80 monoclonal antibody column. Recombinant human RPA was expressed in Escherichia coli and purified by Affigel Blue (Bio-Rad) column chromatography as described ( 30). In Vitro Protein Kinase Assays.Ĭell extracts were prepared as described ( 9) with the exception that 0.5 M NaCl was used to extract the isolated nuclei. To verify the identity of the phosphorylated RPA forms protein extracts were treated with 100 units of λ phosphatase (New England Biolabs) for 0.5 h at 30☌ before electrophoresis. Immunoreactive bands were visualized using the ECL system (Amersham). HCl/ 137 mM NaCl/1% Tween-20/and either 2% BSA or 5% milk) and were incubated with either a p53-specific monoclonal antibody (Ab-1 Oncogene Science) or a polyclonal antibody specific for RPA p34 raised in rabbits against synthetic peptides of the conserved C terminus (SP509).Membranes were then blocked with TBS-T (20 mM Tris After transfer, the gels (both RPA and p53) and the nitrocellulose membranes (p53) were stained with 0.15% Coomassie blue stain and Ponceau S solution (Bio-Rad), respectively, to ensure equal loading and transfer of the protein samples. Proteins were then transferred to Hybond ECL nitrocellulose paper (Amersham) using a semidry apparatus (Bio-Rad) at a maximum of 250 mA/gel and 15 V for 1–1.5 h. Samples (50 μg total protein) were electrophoresed through a 15% (for RPA detection) or 10% (for p53 detection) denaturing SDS/polyacrylamide gel at 50 mA/gel. Whole-cell extracts were prepared as described ( 7). Control cells (0 h time point) were mock-irradiated. SCID, C.B-17, MO59J, and MO59K cells were grown in 100-mm 2 dishes to 50–80% confluence and irradiated at either 6 Gy (for the p53 analysis) or 50 Gy (for RPA phosphorylation) in a cesium irradiator. We conclude that the DNA damage response involving p53 and RPA is not associated with the defect in DNA repair in SCID cells and that the physiological substrate(s) for DNA-PK essential for DNA repair has not yet been identified. These results suggest that DNA-PK is not the only kinase capable of phosphorylating RPA. The hyperphosphorylation of RPA p34 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA in crude extracts derived from both C.B-17 and SCID cells. Our findings indicate that ( i) p53 levels are increased in SCID cells following ionizing radiation, and ( ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. To determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (human DNA-PK cs-deficient glioblastoma) cells with their respective wild-type parents following ionizing radiation. DNA-PK phosphorylates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells to DNA damage. The catalytic subunit of DNA-dependent protein kinase (DNA-PK CS) has previously been identified as a strong candidate for the SCID gene. Severe combined immunodeficient (SCID) mice display an increased sensitivity to ionizing radiation compared with the parental, C.B-17, strain due to a deficiency in DNA double-strand break repair.
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