In Vivo Assembly of Overproduced DNA Polymerase III

Abstract

The genes for the polymerase core (αεθ) 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 α, αε and αεθ 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 106 units/mg in a gap-filling assay. Kinetics studies showed that neither nor θ influenced the K(m) of a for deoxynucleotide triphosphate and only slightly decreased the K(m) of a for DNA, although ε was absolutely required for maximal DNA synthesis. The rate of DNA synthesis by α- reconstituted holoenzyme τ using complex was about 5-fold less than that of αε or αεθ-reconstituted holoenzyme as determined by a gel analysis. The processivity of α-reconstituted holoenzyme was very similar to that of αεθ-reconstituted holoenzyme when τ complex was used as a clamp loader.