The replicative polymerases PolC and DnaE are required for theta replication of the Bacillus subtilis plasmid pBS72

Abstract

Plasmids are the tools of choice for studying bacterial functions involved in DNA maintenance. Here a genetic study on the replication of a novel, low-copy-number, Bacillus subtilis plasmid, pBS72, is reported. The results show that two plasmid elements, the initiator protein RepA and an iteron-containing origin, and at least nine host-encoded replication proteins, the primosomal proteins DnaB, DnaC, DnaD, DnaG and DnaI, the DNA polymerases DnaE and PolC, and the polymerase cofactors DnaN and DnaX, are required for pBS72 replication. On the contrary, the cellular initiators DnaA and PriA, the helicase PcrA and DNA polymerase I are dispensable. From this, it is inferred that pBS72 replication is of the theta type and is initiated by an original mechanism. Indirect evidence suggests that during this process the DnaC helicase might be delivered to the plasmid origin by the weakly active DnaD pathway stimulated by a predicted interaction between DnaC and a domain of RepA homologous to the major DnaC-binding domain of the cellular initiator DnaA. The plasmid pBS72 replication fork appears to require the same functions as the bacterial chromosome and the unrelated plasmid pAMb1. Most importantly, this replication machinery contains the two type C polymerases, PolC and DnaE. As the mechanism of initiation of the three genomes is substantially different, this suggests that both type C polymerases might be required in any Cairns replication in B. subtilis and presumably in other bacteria encoding PolC and DnaE. INTRODUCTIONDNA replication is a ubiquitous biological process carried out by proteins that have evolved profoundly since the last common ancestor. For key elements like initiators, helicases, primases, catalytic DNA polymerases and processivity factors, their evolution has been so extensive that they are postulated to originate from peptides that diverged beyond recognition or from different proteins (Edgell & Doolittle, 1997;Giraldo, 2003;Leipe et al., 1999). Surprisingly, this diversity, obvious when comparing Eubacteria to Archaea and Eukarya, is also manifest among bacterial species. For instance, in the well characterized eubacteria Escherichia coli and Bacillus subtilis, the primosomes required for initiating replication at origins or collapsed replication forks have been shown to greatly differ in their constitution and mechanism of helicase loading (Bruand et al., 1995;Cox et al., 2000;Davey & O'Donnell, 2003;Lemon et al., 2002;Marsin et al., 2001;Polard et al., 2002; Velten et al., 2003). Differences have also been documented in the DNA polymerase III (Pol III) holoenzyme, a subassembly of the replication machinery where DNA synthesis is catalysed. In E. coli this element contains 10 different subunits of which four (h, c, x and y) are missing in B. subtilis (Bruck & O'Donnell, 2000;Bruck et al., 2002;Lemon et al., 2002; MartinezJimenez et al., 2002;McHenry, 2003).Based on systems for which a Pol III holoenzyme has been isolated and/or reconstructed in vitro, it is generally thought that both DNA strands...

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