We intend to recreate the bidirectional replication of p4 DNA in a purified, reconstituted, in vitro reaction. Satellite phage p4 DNA replicates bidirectionally, starting as a double-stranded circular molecule of 11 kb. p4 does not need the host proteins that prime the synthesis of DNA chains (dnaB, dnaG) and must synthesize its own priming apparatus. We observe p4 DNA synthesis in vitro, depending on the p4 Alpha gene product, which we have purified to near-homogeniety. The p4 Alpha protein can prime DNA synthesis on a circular, single-stranded DNA template. We shall characterize the primers made by the p4 Alpha protein, as well as any nuclease and topoisomerase activities that it may have. We shall determine the base sequence of the replication origin and show whether our in vitro replication is origin specific. Bidirectional DNA replication is a key event in the regulation of human cell growth. Understanding of the initiation of DNA replication is therefore essential for the control of normal development, wound-healing and oncogenic transformation.
