PRIMOSOME ASSEMBLY SITE IN BACILLUS-SUBTILIS

A single-strand initiation site was detected on the Enterococcus faeca lis plasmid pAM beta 1 by its ability to prevent accumulation of singl e stranded DNA of a rolling circle plasmid, both in Bacillus subtilis and Staphylococcus aureus, This site, designated ssiA, is located on t he lagging strand template, similar to 150 bp downstream from the repl ication origin. ssiA priming activity requires the DnaE primase, the D naC replication fork helicase, as well as the products of the dnaB, dn aD and dnaI genes of B.subtilis, but not the RNA polymerase, The prima se and the replication fork helicase requirements indicate that ssiA i s a primosome assembly site. Interestingly, the pAM beta 1 lagging str and synthesis is inefficient when any of the proteins involved in ssiA activity is mutated, but occurs efficiently in the absence of ssiA, T his suggests that normal plasmid replication requires primosome assemb ly and that the primosome can assemble not only at ssiA but also elsew here on the plasmid, This work for the first time describes a primosom e in a Gram-positive bacterium, Involvement of the B.subtilis proteins DnaB, DnaD and DnaI, which do not have any known analogue in Escheric hia coli, raises the possibility that primosome assembly and/or functi on in B.subtilis differs from that in E.coli.