THE ORDERED ASSEMBLY OF THE PHI-X174-TYPE PRIMOSOME .3. PRIB FACILITATES COMPLEX-FORMATION BETWEEN PRIA AND DNAT

The properties of two mutant PriA proteins, PriA C439Y and PriA. C445Y have been used to reveal the role of PriB during assembly of the phi X174-type primosome, The replication defects of both mutant PriA prote ins could be rescued by high concentrations of DnaT. Analysis of the f ormation of intermediate complexes in primosome assembly and the effec t of PriB on PriA binding to DNA demonstrated that the mutant PriA pro teins could not form a PriA-PriB complex on DNA carrying a primosome a ssembly site, Consequently, the mutant proteins also could not form Pr iA-PriB-DnaT complexes at concentrations of DnaT sufficient to form su ch a complex with wild-type PriA. In addition, PriB was found to stabi lize wild-type but not mutant PriA proteins on DNA. At high concentrat ions of DnaT, both mutant and wild-type PriA proteins could form a Pri cA-DnaT complex and support PriB-independent phi X174 complementary st rand DNA replication, Thus, during primosome assembly, PriB facilitate s complex formation between PriA and DnaT.