The ordered assembly of the phi X174-type primosome .2. Preservation of primosome composition from assembly through replication

Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated phi X174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, The presence and relative amounts of primosomal proteins in these complexes were determined by Western blotting, Protein-DNA complexes isolated (i) after assembly in the presence of 10 mu M ATP, (ii) after preprimosome movement in the presence of 1 mM ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; preprimosomes contained PriA, PriB, PriC, DnaT, and DnaB, whereas primosomes included DnaG, The stable association of DnaG with the protein-DNA complex could be attributed partially to its ability to remain bound to the primers synthesized, In the absence of PriC, the efficiencies of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a primosome assembly site (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem, 271, 15642-15648), We hypothesize that maintenance of the primosome on the replicated DNA may provide a mechanism to allow primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break repair by permitting the re-establishment of a replication fork.