Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells

Abstract

The virus-coded 72000-Mr DNA-binding protein from adenovirus-type-2-infected cells has been purified to homogeneity by DEAE-cellulose chromatography, selective precipitation and gel filtration. The 72000-Mr DNA-binding protein is phosphorylated and the phosphate is covalently linked predominantly to serine. Analysis of tryptic digests of the 32P-labeled 72000-Mr protein showed that the phosphate residue(s) is present in only one peptide. The DNA-binding fraction contains an additional non-phosphorylated protein with an approximate molecular weight of 45000. Tryptic peptide maps of [35S]methionine-labeled 72000-Mr and 45000-Mr polypeptides are indistinguishable. The amino acid compositions of the 72000-Mr and 45000-Mr polypeptides show closely related distributions. An antiserum produced against the purified 72000-Mr DNA-binding protein precipitates both the 72000-Mr and the 45000-Mr 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.