The genes for the polymerase core (alphaepsilontheta) of the DNA polymerase III holoenzyme map to widely separated loci on the Escherichia coli chromosome. To enable efficient overproduction and in vivo assembly of DNA polymerase III core, artificial operons containing the three structural genes, dnaE, dnaQ, and holE, were placed in an expression plasmid. The proteins alpha, alphaepsilon and alphaepsilontheta were overexpressed and assembled in E. coli and purified to homogeneity. The three purified polymerases had a similar specific activity of about 6.0 x 10(6) units/mg in a gap-filling assay. Kinetics studies showed that neither epsilon nor theta influenced the Km of alpha for deoxynucleotide triphosphate and only slightly decreased the Km of alpha for DNA, although epsilon was absolutely required for maximal DNA synthesis. The rate of DNA synthesis by alpha-reconstituted holoenzyme using tau complex was about 5-fold less than that of alphaepsilon or alphaepsilontheta-reconstituted holoenzyme as determined by a gel analysis. The processivity of alpha-reconstituted holoenzyme was very similar to that of alphaepsilontheta-reconstituted holoenzyme when tau complex was used as a clamp loader.