Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells.
Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation.
D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000502:physiology*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000502:physiology*; D014779:Virus Replication
D004268:DNA-Binding Proteins; D011498:Protein Precursors; D014764:Viral Proteins; D003843:Deoxycytidine Monophosphate; D004265:DNA Helicases