Function of adenovirus terminal protein in the initiation of DNA replication.
An early event in the initiation of adenovirus DNA replication is the formation of a covalent complex between the 87,000-dalton adenovirus terminal protein precursor and 5'- dCMP (pTP-dCMP complex). Nuclear extracts prepared from adenovirus-infected HeLa cells catalyzed complex formation in the presence of ATP, Mg2+, and adenovirus DNA-protein complex but were not active when Pronase-treated DNA was used as template. The activity has been partially purified by chromatography on denatured DNA-cellulose and used to examine whether the 55,000-dalton terminal protein on adenovirus DNA is required for pTP-dCMP complex formation. Results obtained with either DNA-protein complex or Pronase-treated DNA were identical to those obtained using crude nuclear extracts. However, after treatment with piperidine to remove residual peptides. Pronase-treated DNA supported complex formation with the partially purified activity but not with the crude extracts. In addition, when a plasmid containing an origin of adenovirus DNA replication was used as template, the pTP-dCMP complex was formed provided the plasmid was linearized in such a way that the origin was located at the end of the molecule. Neither linearized plasmid DNA with an internal origin nor supercoiled plasmid DNA supported complex formation. Furthermore, after heat denaturation, the linear plasmid DNA still supported complex formation, again provided that the origin was located at the end of the molecule. The partially purified protein fraction supported a limited amount of DNA chain elongation, which permitted exact positioning of the initiation site. These results suggest that enzymes responsible for complex formation recognize a DNA sequence at the origin and that the terminal protein on the template DNA plays a subordinate role.
D000256:Adenoviridae / Q000235:genetics*; D003568:Cytidine Monophosphate / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D010454:Peptide Termination Factors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids
D004279:DNA, Viral; D010454:Peptide Termination Factors; D003568:Cytidine Monophosphate