Viruses are very simple entities. In its most simple form, the virus consists of a protein shell containing genetic material. To propagate the virus needs a host to parasitize, in which it can replicate its genetic material and assembly new virus particles. Viruses that parasitize bacteria are termed bacteriophages. In this study the DNA replication of bacteriophage P2 has been investigated. Bacteriophage P2 replicates by a rolling circle mechanism, which is a replication strategy used by many replicons, ranging from bacterial plasmids to animal viruses. Two phage encoded proteins are necessary for P2 rolling circle replication; A and B. The A protein initiates rolling circle replication by a single-stranded, site-specific nick at the origin of replication. This exposes a free 3' end which serves as a primer for DNA polymerisation. Upon nicking, P2 A links itself covalently to the 5' end of the DNA through one of its two active-site tyrosine residues. The A protein can also catalyse the reverse reaction. This event releases the displaced strand simultaneously as a new round of replication is initiated. The B protein has been shown to be required for lagging strand synthesis and has been suggested to be an E. coli DnaB helicase loader.

In this work, the interplay between the two tyrosine residues located in the presumed catalytic site of P2 A has been closely examined. We found that the two tyrosine residues, 450 and 454, play non-equivalent functional roles in their catalytic activity. We propose that tyrosine 454 initiates replication and that tyrosine 450 only cleaves when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication. Also, data from the catalytic activity of P2 A, involving different divalent cations, suggests that conformational changes occur within the protein upon binding DNA. Another intriguing phenomenon of P2 A is that it acts in cis in vivo, i.e. it can only act upon the molecule from which it was transcribed. Here, we show that the cis-activity is retained in vitro during coupled transcription and translation in a S30 extract and that the information required for the cis-activity in vivo and in vitro is contained entirely in the coding region for the A protein. An elegant model for the mechanism of the P2 A cis-activity is also presented. Further, in this work in vivo and in vitro evidence for a physical association between P2 B and E. coli DnaB are provided supporting the view that P2 B is a helicase loader. Presumably, the B protein plays an important role as a molecular matchmaker for the whole P2 replisome assembly. Here, also presented are the enigma of the inverted palindrome nature of the P2 origin, the P2 A localisation within the bacterial cell, other RCR initiating enzymes, a compilation of all known cis-acting proteins, evolutionary aspects of cis-action, the outline for exploiting P2 A as a biotechnology tool and much much more.