Enzyme Studies of ϕX174 DNA Replication

This chapter describes the enzyme studies of ϕx174 DNA rep1ication. Replication of the single-stranded DNA of phage ϕx174 can be divided into three stages. In stage I, the single-stranded circle is converted into a duplex replicative form (RF-I) by the action of the DNA replication enzymes of the Escherichia coli host cell. In the second stage, multiple copies of RF-I are generated using the newly synthesized RF-I as a template; this process requires two additional proteins, the phage-encoded gene A protein and host rep protein. In the final stage, further production of RF-I ceases and only the viral strand is synthesized for envelopment into the capsid of a progeny phage. This last stage requires the gene A, rep proteins and also the products of the phage genes B, D, F, G, and H. The enzymology of E. coli replication proteins employed in ϕxDNA replication is a guide to their action in replicating the host chromosome. The chapter discusses the six repriming proteins that are required for SS––>RF on SSB-Coated ϕX DNA. It also presents that protein n’ recognizes a specific sequence of ϕX DNA. The chapter concludes with reconstitution of the RF ­––>RF system from purified proteins.