In vivo assembly of overproduced DNA polymerase III - Overproduction, purification, and characterization of the alpha, alpha-epsilon, and alpha-epsilon-theta subunits

The genes for the polymerase core (alpha epsilon theta) 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, alpha epsilon and alpha epsilon theta 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 K-m of alpha for deoxynucleotide triphosphate and only slightly decreased the K-m 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 alpha epsilon or alpha epsilon theta-reconstituted holoenzyme as determined by a gel analysis. The processivity of alpha-reconstituted holoenzyme was very similar to that of alpha epsilon theta-reconstituted holoenzyme when tau complex was used as a clamp loader.