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.