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.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, ⌽29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from ⌽29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3 3 5 exonuclease domain (DNA polymerase II exo ؊ and T7 pol exo ؊ ) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.Misalignment of two DNA strands during replication can lead to DNA rearrangements such as deletions or duplications of varying lengths ranging from several nucleotides to entire genes. This process, designated replication slippage (as well as copy-choice recombination), has been suspected for a long time to occur both in prokaryotes and eukaryotes between repeated DNA sequences. The process is thought to encompass the following steps: (i) copying of the first duplication by the replication machinery, (ii) replication pausing and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second duplication, and (iv) resumption of the DNA synthesis. A heteroduplex is thus formed, containing one parental and one recombinant strand, which are separated by a second round of replication.Replication slippage has been widely proposed as a probable mechanism of genome rearrangements, such as deletions between short duplications in bacteria (1-3), yeast (4), and mammalian mitochondria (5) or deletions between long tandem repeats in Escherichia coli (6 -8), as well as microsatellite instability (for reviews see Refs. 9 -12). Direct evidence for the slippage has been obtained in vivo, in E. coli (13), and in vitro (14). In the latter study, it was shown that E. coli DNA polymerase III holoenzyme (pol III HE), 1 the enzyme that replicates the cell chromosome (for review see Ref. 15), was able to slip, which is of particular significance in vie...

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...

No abstract

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of27 bp that flank an inverted repeat of '300 bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the 3 subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.