Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.
Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.
Kusukawa
J
J
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421.
Ramachandra
M
M
Nakano
R
R
Padmanabhan
R
R
D000260:Adenoviruses, Human / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014357:Trypsin; D014764:Viral Proteins
D010710:Phosphates; D010748:Phosphopeptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011498:Protein Precursors; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; C085476:pTP protein, adenovirus; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin