THE ORDERED ASSEMBLY OF THE PHI-X174-TYPE PRIMOSOME .1. ISOLATION ANDIDENTIFICATION OF INTERMEDIATE PROTEIN-DNA COMPLEXES

The phi X-type primosome was discovered during the resolution and reco nstitution in vitro of the complementary strand DNA replication step o f the phi X174 viral life cycle, This multienzyme bidirectional helica se-primase complex can provide the DNA unwinding and Okazaki fragment- priming functions at the replication fork and has been implicated in c ellular DNA replication, repair, and recombination. We have used gel m obility shift assays and enhanced chemiluminescence Western analysis t o isolate and identify the pathway of primosome assembly at a primosom e assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fra gment. The first three steps do not require ATP and are as follows: (i ) PriA recognition and binding to the PAS, (ii) stabilization of the P riA-PAS complex by the addition of PriB, and (iii) formation of a PriA -PriB-DnaT-PAS complex. Subsequent formation of the preprimosome invol ves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex. The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC. A transient interaction betwe en the preprimosome and DnaG generates the five-protein primosome. As described in an accompanying article (Ng, J. Y., and Marians, K. J. (1 996) J. Biol. Chem. 271, 15649-15655), when assembled on intact phi X1 74 phage DNA, the primosome also contains PriC.