DNAX COMPLEX OF ESCHERICHIA-COLI DNA-POLYMERASE-III HOLOENZYME - CENTRAL ROLE OF TAU IN INITIATION COMPLEX ASSEMBLY AND IN DETERMINING THE FUNCTIONAL ASYMMETRY OF HOLOENZYME

The alternative forms of the DnaX protein found in Escherichia coli DN A polymerase III holoenzyme, tau and gamma, were purified from extract s of strains carrying overexpressing plasmids mutated in their framesh ifting sequences such that they produced only one subunit or the other . The purified subunits were used to reconstitute the tau and gamma co mplexes which were characterized by functional assays. The gamma compl ex-reconstituted holoenzyme required a stoichiometric excess of DNA po lymerase III core, beyond physiological levels, for activity. The gamm a subunit stimulated the gamma complex 2-fold, but could not be used t o reconstitute a holoenzyme with gamma complex and stoichiometric quan tities of core. In the presence of adenosine 5'-O-(3'-thiotriphospate) (ATP gamma S), the DNA polymerase III holoenzyme behaves as an asymme tric dimer; it can form only initiation complexes with primed DNA in o ne-half of the enzyme (Johanson, K. O., and McHenry, C. S. (1984) J. B iol. Chem. 259, 4589-4595). An asymmetric distribution of two products of the dnaX gene, gamma and tau has been postulated to underlie the a symmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the gamma or tau DnaX prot eins. We observed that, although gamma could function in the presence of ATP and high concentrations of DNA polymerase III core, it was near ly inert in the presence of ATP gamma S. In contrast, tau-containing h oloenzyme behaved exactly like native holoenzyme in the presence of AT P gamma S. These results implicate tau as a key component required to reconstitute holoenzyme with native behavior and show that tau plays a key role in initiation complex formation. These results also show tha t gamma is not a necessary component, since all of the known propertie s of native holoenzyme can be reproduced with a 9-subunit tau-holoenzy me.