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DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III-clamp-exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.

DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands. In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair. MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood. We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis. A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry. When mutated, the asymmetry is lost and mismatch repair in vivo is impaired. We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.