The Mutant bE202K Sliding Clamp Protein Impairs DNA Polymerase III Replication Activity

Expression of the Escherichia coli dnaN-encoded b clamp at $10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess b clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability 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 bE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIa and Pol III core (Pol IIIa«u ), suggesting that it could still sequester Pol III effectively. Of interest, bE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant bE202K 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 the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the b clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The b 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 b clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant b clamps that fail to inhibit growth. Their analysis revealed that bE202K is unique among them. Our work offers new insights into how the b clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV.