Formation in vitro of the pTP-dCMP initiation complex of human adenovirus type 12

We report the covalent addition of [32P]dCMP to a protein from group A adenovirus 12 (Ad12)-infected human (KB) cells in vitro, using crude extracts. Synthesis of the 60K protein-dCMP complex required a DNA template containing a terminally located adenovirus replication origin; the protein-dCMP bond was alkali-labile but acid-stable. We therefore conclude that this product is the Ad12 terminal protein precursor (pTP)-dCMP initiation complex for DNA replication. Synthesis of Ad12 pTP-dCMP was specific for dCTP but was stimulated by dATP. In contrast to Ad2, the Ad12 initiation reaction required ATP. Antipeptide antiserum targeted to Ad DNA polymerase inhibited Ad12 pTP-dCMP synthesis in vitro, providing evidence that Ad DNA polymerase catalyse dCMP addition to pTP during initiation.