Purification and Characterization of the Phosphorylated DNA-Binding Protein from Adenovirus-Type-2-Infected Cells

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Abstract
The virus-coded 72,000-MW DNA-binding protein from adenovirus-type-2-infected [human cervical carcinoma HeLa] cells was purified to homogeneity by DEAE-cellulose chromatography, selective precipitation and gel filtration. The 72,000-MW DNA-binding protein is phosphorylated and the phosphate is covalently linked predominantly to serine. Analysis of tryptic digests of the 32P-labeled 72,000-MW protein showed that the phosphate residue(s) is present in only 1 peptide. The DNA-binding fraction contains an additional non-phosphorylated protein with an approximate MW of 45,000. Tryptic peptide maps of [35S]methionine-labeled 72,000-MW and 45,000-MW polypeptides are indistinguishable. The amino acid compositions of the 72,000-MW and 45,000-MW polypeptides show closely related distributions. An antiserum produced against the purified 72,000-MW DNA-binding protein precipitates both the 72,000-MW and the 45,000-MW protein from extracts of adenovirus-infected cells. Immunofluorescence studies revealed DNA-binding protein to be accumulated in characteristic structures in nuclei of the infected cells.