Expression of the
E. coli
dnaN
-encoded β clamp at ≥10-fold higher than chromosomally-expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we measured the ability of eight mutant clamps obtained by their inability to impede growth to stimulate Pol III replication
in vitro
. Compared with the wild type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β
E202K
mutant, which bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still effectively sequester Pol III. Of interest, β
E202K
supported
in vitro
DNA replication by Pol II and Pol IV, but was defective with Pol III. Genetic experiments indicated that the
dnaN
E202K
strain remained proficient in DNA damage-induced mutagenesis, but was modestly induced for SOS and displayed sensitivity to ultraviolet light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β
E202K
clamp to support Pol III replication
in vivo
with its
in vitro
defect in DNA replication. Taken together, our results: (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for existence of an additional mechanism that contributes to lethality and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than currently appreciated.
IMPORTANCE
The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners, and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes
E. coli
growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β
E202K
is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of
E. coli
DNA polymerases II, III and IV.
The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an “Ajar” Intermediate Conformation in the Nucleotide Selection Mechanism