A study of the in vitro initiation of adenovirus DNA replication

Abstract

The development of systems in which adenovirus DNA can be replicated in vitro has led to the elucidation of the sequences essential for origin function and to the identification of the proteins required for viral DNA replication. Much of the information currently available has been derived from investigations carried out using adenovirus types 2 and 5 which in addition to the viral proteins, adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, require cellular proteins nuclear factor I and nuclear factor III for efficient initiation of DNA replication. In contrast adenovirus type 4 replicates Its DNA efficiently without these cellular proteins. Correspondingly its minimal origin of replication is remarkably simple in structure, consisting of only the terminal 18bp of the adenovirus genome ('the core sequence'). The effect of point mutations in the core region on adenovirus type 4 DNA replication in vitro was investigated and it was found that mutations within two discrete domains had a married deleterious effect on initiation of DNA replication. The crude Ad4 infected cell extracts initially used for in vitro DNA replication were fractionated and it was found that only four detectable proteins, three of which were identified as viral DNA polymerase, precursor terminal protein and DNA binding protein gave efficient DNA replication in vitro and furthermore behaved similarly to unfractionated infected cell extracts in the presence of template which contained point mutations. To examine a possible role of the core region of the origin as containing sites for specific interactions with viral replication proteins, purified adenovirus type 5 precursor terminal protein and DNA polymerase were assayed for their ability to recognise the terminal 1-18 sequence. It was found that both proteins independently and as a heterodimer bound specifically to a sequence corresponding to the core origin of replication, suggesting that sequences within this region are important for localisation of DNA replication proteins at the origin via a sequence specific DNA-protein interaction.