IN-VIVO ASSEMBLY OF OVERPRODUCED DNA-POLYMERASE-III - OVERPRODUCTION,PURIFICATION, AND CHARACTERIZATION OF THE ALPHA-SUBUNIT, ALPHA-EPSILON-SUBUNIT, AND ALPHA-EPSILON-THETA-SUBUNIT

The genes for the polymerase core (alpha epsilon theta) of the DNA pol ymerase III holoenzyme map to widely separated loci on the Escherichia coil chromosome. To enable efficient overproduction and in vivo assem bly of DNA polymerase III core, artificial operons containing the thre e structural genes, dnaE, dnaQ, and holE, were placed in an expression plasmid. The proteins alpha, alpha epsilon and alpha epsilon theta we re overexpressed and assembled in E. coil and purified to homogeneity. The three purified polymerases had a similar specific activity of abo ut 6.0 x 10(6) units/mg in a gap-filling assay. Kinetics studies showe d that neither epsilon nor theta influenced the K-m of alpha for deoxy nucleotide triphosphate and only slightly decreased the K-m of alpha f or DNA, although epsilon was absolutely required for maximal DNA synth esis. The rate of DNA synthesis by alpha-reconstituted holoenzyme usin g tau complex was about 5-fold less than that of alpha epsilon or alph a epsilon theta-reconstituted holoenzyme as determined by a gel analys is. The processivity of alpha-reconstituted holoenzyme was very simila r to that of alpha epsilon theta-reconstituted holoenzyme when tau com plex was used as a clamp loader.