In vivo protein interactions within the Escherichia coli DNA polymerase III core.


The mechanisms that control the fidelity of DNA replication are being ...
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 versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389-4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (alpha subunit [possessing DNA polymerase activity]) and DnaQ (epsilon subunit [possessing 3'-->5' exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (theta subunit) proteins. We also tested the interactions of the 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 mutations do not affect the epsilon subunit-alpha subunit and epsilon subunit-theta subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the epsilon subunit with both the alpha and the theta subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the epsilon protein, which leads to altered interactions within the Pol III core.




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