Figure 5. Investigation of the functional interactions between the subunits of the αβ₂‘ε’ complex of Mtb DNA pol III.

(a) Primer-extension assay of different ratios of α and ‘ε’ subunits using a primed dsDNA substrate. Mixtures of α and ‘ε’ subunits exhibit unexpectedly high exonuclease activity on paired duplex DNA and no polymerase activity in vitro. Increasing amounts of the α subunit fail to weaken the exonuclease activity of the ‘ε’ subunit in vitro. (b) Exonuclease assay of the ‘ε’ mutant (‘ε’^exo-) using a ssDNA substrate. Mutation of the active site of the ‘ε’ subunit (D20A, E22A and D104A) successfully destroys its exonuclease activity. (c) Primer-extension and exonuclease assays of the α subunit with increasing amounts of ‘ε’^-exo using a primed dsDNA substrate. The ‘ε’^-exo mutant modestly enhances the polymerase activity of the α subunit (left panel), but has no effect on its exonuclease activity (right panel). (d) Primer-extension and exonuclease assays of the α subunit with increasing amounts of β₂ clamp using a primed dsDNA substrate. While the polymerase activity of the α subunit is strongly promoted by the presence of the β₂ clamp (left panel), its exonuclease activity is only slightly increased (right panel). 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 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.