Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro.
Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 37 degrees C in vitro. These extracts were completely complemented by the addition of wild-type adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates.
D000260:Adenoviruses, Human / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D005816:Genetic Complementation Test; D006367:HeLa Cells; D008970:Molecular Weight; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors; D014779:Virus Replication
D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D004265:DNA Helicases