VIVO PROTEIN INTERACTIONS WITHIN THE ESCHERICHIA-COLI DNA-POLYMERASE-III CORE

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versi ons of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA po lymerase III (Pol III) of Escherichia coli could be essential for main taining the optimal fidelity of DNA replication (H. Maki and A. Kornbe rg, Proc. Natl. Acad. Sci. USA 84:4389-4392, 1987). We have been parti cularly interested in elucidating the physiological role of the intera ctions between the DnaE (alpha subunit [possessing DNA polymerase acti vity]) and DnaQ (epsilon subunit [possessing 3'-->5' exonucleolytic pr oofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze s pecific in vivo protein interactions. In this report, we demonstrate i nteractions between the DnaE and DnaQ proteins and between the DnaQ an d HolE (theta subunit) proteins. We also tested the interactions of th e wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutatio ns do not affect the epsilon subunit-alpha subunit and epsilon subunit -theta subunit interactions. However, the dnaQ49 mutation greatly redu ces the strength of interaction of the epsilon subunit with both the o r and the theta subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the epsilon protein, which l eads to altered interactions within the Pol III core.