Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Abstract

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...

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