Primosome assembly site in Bacillus subtilis.

Abstract

Corresponding author A single-strand initiation site was detected on the Enterococcus faecalis plasmid pAMP1 by its ability to prevent accumulation of single stranded DNA of a rolling circle plasmid, both in Bacillus subtilis and Staphylococcus aureus. This site, designated ssiA, is located on the lagging strand template, -150 bp downstream from the replication origin. ssiA priming activity requires the DnaE primase, the DnaC replication fork helicase, as well as the products of the dnaB, dnaD and dnal genes of B.subtilis, but not the RNA polymerase. The primase and the replication fork helicase requirements indicate that ssiA is a primosome assembly site.Interestingly, the pAM01 lagging strand synthesis is inefficient when any of the proteins involved in ssiA activity is mutated, but occurs efficiently in the absence of ssiA. This suggests that normal plasmid replication requires primosome assembly and that the primosome can assemble not only at ssiA but also elsewhere on the plasmid. This work for the first time describes a primosome in a Gram-positive bacterium. Involvement of the B.subtilis proteins DnaB, DnaD and DnaI, which do not have any known analogue in Escherichia coli, raises the possibility that primosome assembly and/or function in B.subtilis differs from that in E.coli.

Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid‐encoded resolution system

“…Such a switch may take place in plasmids of the pAM␤1 and ColE1 families because they all carry a primosome-assembly site on the lagging-strand template, immediately upstream of a protein-binding site (Nomura and Ray, 1980;Marians et al, 1982;Nijkamp et al, 1986;Morlon et al, 1988;Boyd et al, 1989;Brantl et al, 1990;Pujol et al, 1994;Bruand et al, 1995;Fu et al, 1995). The arrested D-loop intermediates that may form in these plasmids through the arrest of Pol I at a Res-res or Mob-bom roadblock could therefore be the substrate for Pol I to Pol III HE switching and for replisome entry.…”

“…1, binding of Res␤ to a non-replicating molecule; 2, initiation of leading-strand synthesis by Pol I; 3, arrest of Pol I progression by collision with the Res␤-DNA complex, release of Pol I, ssiA activation and primosome assembly; 4, release of Res␤ by a helicase, primer synthesis on the laggingstrand template by the primase, Pol III HE assembly at the 3Ј ends of the primer and nascent leading strand; 5, concerted synthesis of the leading and lagging strands by the replisome. Bruand et al, 1995) and E. coli DH5␣ (Sambrook et al, 1989). Induction of competence, transformation and growth conditions were as previously described (Janniè re and Ehrlich, 1987).…”

“…Second, the presence of a palindrome 100 bp long close to and downstream of the replication origin leads to the appearance of aberrant replication products formed by a template strand switch of Pol I (Bruand et al, 1993; the process was termed 'snap-back' replication by Backman et al, 1978). Third, the bulk of DNA synthesis of pAM␤1 and related plasmids is inhibited at restrictive temperatures in thermosensitive mutant strains impaired in either different subunits of Pol III HE or in the replication fork helicase and in several primosomal proteins (Ceglowski et al, 1993;Bruand et al, 1995).…”

“…From this site, DNA replication progresses unidirectionally in the direction of repE transcription (Bruand et al, 1991). An additional, non-essential element is a primosomeassembly site, ssiA, located on the lagging-strand template Ϸ150 bp downstream of ori (Bruand et al, 1995). This signal is thought to stimulate replisome assembly when it is rendered single stranded in replication intermediates carrying D-loop structures (Bruand et al, 1995).…”

“…An additional, non-essential element is a primosomeassembly site, ssiA, located on the lagging-strand template Ϸ150 bp downstream of ori (Bruand et al, 1995). This signal is thought to stimulate replisome assembly when it is rendered single stranded in replication intermediates carrying D-loop structures (Bruand et al, 1995). pAM␤1 encodes a resolution system which maintains plasmid population in the monomeric state (Swinfield et al, 1991).…”