Journal: Nucleic Acids Research
Article Title: Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage
doi: 10.1093/nar/gkr647
Figure Lengend Snippet: Identification of DNA-PK phosphorylation sites in PNKP. ( A ) Purified His-tagged PNKP (0.5 µg) was incubated with or without DNA-PK (400 ng), calf thymus DNA (10 µg/ml) or 10 µM wortmannin, as indicated by the + or − symbols. The reaction was carried out at 30°C for 30 min and stopped by boiling in SDS sample buffer. Proteins were separated by electrophoresis on a 10% SDS polyacrylamide gel stained with Coomassie blue, and dried for autoradiography. ( B ) Purified PNKP, phosphorylated by DNA-PK, was separated by electrophoresis on an SDS polyacrylamide gel and then digested with trypsin as described in ‘Materials and methods’ section. The resulting 32 P-labeled peptides were chromatographed on a Vydac 218TP54 C 18 column (Separation Group, Hesperia, CA, USA) equilibrated in 0.1% (v/v) trifluoroacetic acid in water. The column was developed with a linear acetonitrile gradient (dashed diagonal line, right hand Y -axis) at 0.8 ml/min, and fractions of 0.4 ml were collected: >80% of the radioactivity applied to the column was recovered in the fractions. ( C ) An aliquot of the major 32 P-labeled peptide derived from phosphorylated PNKP ( B) was covalently coupled to a Sequelon acrylamide membrane and analyzed on an Applied Biosystems 476A sequenator. 32 P radioactivity was measured after each cycle of Edman degradation. In combination with MALDI-TOF MS, database searching against predicted PNKP tryptic peptides and phosphoamino acid analysis (data not shown) enabled the identification of the site of phosphorylation in the peptide. The putative amino acid sequence of this peptide, deduced from a combination of phosphoamino acid analysis, MS and solid-phase sequencing, is indicated at the bottom of the graph.
Article Snippet: From the reaction mixture, 4 μl samples were taken at several incubation times up to 10 min and mixed with 2 μl of sequencing gel loading dye (Fisher Scientific), boiled and run at 200 V on a 12% polyacrylamide sequencing gel (Bio-Rad) containing 7 M urea (EMD).
Techniques: Phospho-proteomics, Purification, Incubation, Electrophoresis, Staining, Autoradiography, Labeling, Radioactivity, Derivative Assay, Membrane, Phosphoamino Acid Analysis, Sequencing
