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.

In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

“…Rabbit polyclonals directed against (1) SRm160 (1/250, Blencowe et al 1998); (2) (Linné et al 1977). Mouse monoclonals directed against (1) Sm antigen of SnRNPs (mAb Y12; Lerner et al 1981); (2) U2 snRNP specific protein BЈЈ (mAb 4G3; Habets et al 1989).…”

The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies.

“…The following antibodies were used: affinity-purified rabbit antibodies against cyclin H (Shiekhattar et al 1995), mouse monoclonal MO-1.1 against cdk7 , mouse monoclonal 3C9 against p62 (Fischer et al 1992), rabbit antiserum against MAT1 (Tassan et al, 1995), rabbit antiserum 204.3 against human p80-coilin (Bohmann et al 1995a), mouse monoclonal 1D4-8 against human p80-coilin (Rebelo et al 1996), monoclonal antibody 8H7 against the human XPG endonuclease (O'Donovan et al 1994), monoclonal antibody PC10 against proliferating cell nuclear antigen (PCNA; Dako, Dakopatts, Glostrup, Denmark), and rabbit serum directed against the adenoviral protein DNA-binding protein (DBP; Linne et al, 1977).…”

Phosphorylation of Adenovirus DNA-binding Protein

“…Previous experiments (Linne et al, 1977;Russell & Blair, 1977) had established that during the course of Ad infection a sequence of phosphorylation events occurred in the infected cells, as observed by SDS-PAGE followed by autoradiography. A prominent early event was the phosphorylation of DBP, followed at later times of infection by other phosphorylations such as that of the virus-encoded 100K polypeptide.…”