Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases.

摘要

The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.