DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis. Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE(BS), which is homologous to dnaE. We show that, in B. subtilis, dnaE(BS) is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B. subtilis, as was previously observed in eukaryotes. dnaE(BS) appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B. subtilis and in many other bacteria that contain both polC and dnaE genes.

Plasmid pAMfil from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAMI31 minimal replicon shares no homology with highly conserved ColEl-type replicons, which use Poll for initiation but do not encode a Rep, or with CoIE2 and CoIE3 replicons, which require Poll for replication and encode a Rep. We propose that pAM,B1 and a number of other naturally occurring and closely related plasmids form a distinct plasmid class.Circular bacterial plasmids use two modes of DNA replication, known as rolling circle and theta. The former, first found for single-stranded DNA phages of Escherichia coli, is frequently used by small (< 10-kb) plasmids from Gram-positive bacteria (ref.

BackgroundA challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions.Methodology/Principal FindingsWe report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature. Full restoration of viability by such alterations was limited to cells with mutations in three elongation factors (the lagging strand DnaE polymerase, the primase and the helicase) out of a large set of thermosensitive mutants affected in most of the replication proteins. Restoration of viability resulted, at least in part, from maintenance of replication protein activity at high temperature. Physiological studies suggested that this restoration depended on the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway and occurred in both glycolytic and gluconeogenic regimens. Restoration took place abruptly over a narrow range of expression of genes in the three-carbon part of glycolysis. However, the absolute value of this range varied greatly with the allele in question. Finally, restoration of cell viability did not appear to be the result of a decrease in growth rate or an induction of major stress responses.Conclusions/SignificanceOur findings provide the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed. It is proposed that related systems are ubiquitous.

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.

In a large group of organisms including low G ؉ C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5-DNA end. DnaE is devoid of 3 3 5-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.In all living organisms, DNA replication is carried out by a functionally highly conserved protein complex. Genetic and biochemical data have shown that this complex, called DNA polymerase holoenzyme, contains two copies of an essential replicative DNA polymerase in Escherichia coli, T4 and T7 phages, and SV40 (reviewed in Refs. 1-6). In contrast, replication requires two different polymerases in bacteria Bacillus subtilis and Staphylococcus aureus (pol 1 C and DnaE, C family (7, 8)) and in eukaryotes including Saccharomyces cerevisiae, Xenopus, and human (pol ␦ and pol ⑀, B family; reviewed in Refs. 5 and 9 -11). Thus, holoenzyme of these organisms might be more complex, containing two different polymerases instead of two copies of a single polymerase. This higher level of complexity would hold true for many organisms as follows: (i) systematic sequencing of bacterial genomes (more than 100 completed to date) revealed that ϳ50% carry at least two copies of dnaE or contain dnaE and polC (no genome containing only polC has been detected so far), and (ii) pol ␦ and pol ⑀ seem to be ubiquitous in eukaryotes. It is well established that pol C in bacteria and pol ␦ in eukaryotes are required at the replication fork (5, 9 -15). On the other hand, the specific roles of DnaE and pol ⑀ during replication are still not known. In B. subtilis, it was reported that the purified DnaE protein has a DNA polymerase activity devoid of proofreading activity and presents a high affinity for dNTP (14,16). Genetic and cytological data as well as in vivo assays of radioactive precursor incorporation have shown that DnaE, like pol C, is essential for the elongation phase of replication and is associated with the replication factory at mid-cell (7). Moreover, stu...