Figure 5

Restoration of metal-binding in E. coli Pol III does not induce exonuclease activity. (A) E. coli Pol III wild-type, 3mPHP and 4mPHP mutants show virtually no exonuclease activity in our measurements, as opposed to E. coli Pol III ϵ subunit that shows very robust activity under the same experimental conditions. The 5mPHP mutant shows some exonuclease activity, that is ~30-fold lower than that of the E. coli Pol III ϵ subunit. However, the metal-dependence of this activity is identical to that of the ϵ subunit. For both protein preparations, the exonuclease activity is stimulated by Mg²⁺ (B) and Mn²⁺, but is inhibited by Zn²⁺ (C; 0.3 mM MnCl₂ background), suggesting that the observed activity for the 5mPHP preparation is due to contamination by ϵ subunit.