Figure 6. Role of the β₂ clamp in the αβ₂‘ε’ complex of Mtb DNA pol III.

(a) Primer-extension assay of the αβ₂‘ε’ replicase. The β₂ clamp strongly promotes the polymerization and reduces the exonuclease activity of the αβ₂‘ε’ complex. (b) Primer-extension assay of the αβ₂‘ε’^exo- replicase. Once the exonuclease activity of the ‘ε’ subunit is mutated, the β₂ clamp promotes the polymerase activity, and very slightly enhances the exonuclease activity of the αβ₂‘ε’^exo- replicase. (c) Exonuclease assays of the αβ₂‘ε’ replicase. Increasing the amount of the β₂ clamp does not reduce the exonuclease activity of the αβ₂‘ε’ complex in the absence of the 4 dNTPs. (d) Exonuclease assays of different ratios of ‘ε’ and the β₂ clamp. Increasing the amount of the β₂ clamp fails to directly preclude the exonuclease activity of the ‘ε’ subunit in vitro. (e) Primer-extension assay of Mtb DNA pol III subassemblies. Equimolar mixtures of the α and ‘ε’ subunits exhibited only exonuclease activity, both in the presence (holoenzyme) and absence (αβ₂‘ε’) of the clamp loader and SSB, and the presence of the β₂ clamp reduced exonuclease activity and restored polymerase activity. The reaction conditions of the polymerase (PA) and exonuclease assays (EA) were the same, except that the 4 dNTPs were omitted in the exonuclease assay. The primed dsDNA substrate used in all the above experiments was produced by annealing a 5′-³²P-labeled ssDNA primer (20-mer) to an unlabeled ssDNA template (40-mer). Results presented are representative of at least three replicate experiments.