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J Bacteriol, March 1998, p. 1563-1566, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

In Vivo Protein Interactions within the Escherichia coli DNA Polymerase III Core

Piotr Jonczyk,1,* Adrianna Nowicka,1 Iwona J. Fijalkowska,1 Roel M. Schaaper,2 and Zygmunt Ciesla1

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland,1 and Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 277092

Received 13 August 1997/Accepted 5 January 1998

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 (varepsilon  subunit [possessing 3'right-arrow5' 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 varepsilon  subunit-alpha subunit and varepsilon  subunit-theta subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the varepsilon  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 varepsilon  protein, which leads to altered interactions within the Pol III core.

* Corresponding author. Mailing address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland. Phone and fax: (48)39 12 16 23. E-mail: piotrekj{at}ibbrain.ibb.waw.pl.




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