Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When Xba I digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments...

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Bibliographic Details
Published in:Nucleic acids research 1983-04, Vol.11 (7), p.1975-1989
Main Authors: Bergen, B. G. M. van, Ley, P.A. van der, Driel, W. van, Mansfeld, A. D. M. van, Vliet, P. C. van der
Format: Article
Language:English
Subjects:
Adenovirus
Adenoviruses, Human - genetics
Base Composition
Base Sequence
Cloning, Molecular
DNA Replication
DNA Restriction Enzymes
DNA, Viral - genetics
HeLa Cells - metabolism
Humans
Plasmids
Templates, Genetic
Viral Proteins - genetics
Virus Replication
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Summary:Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When Xba I digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9–22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4–8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/11.7.1975