Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli

Abstract

Background: A strong mutator mutation, dnaE173, leads to a Glu612 → Lys amino acid change in the α subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3′ → 5′ exonuclease activity of the ε subunit. The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the α subunit is hampered by the dnaE173 mutation. Results: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the β clamp subunit) and the β subunit showed a strong resistance to replication-pausing on the template DNA and readily promoted strand-displacement DNA synthesis. Unlike wild-type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the β-clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one-third of that determined for the wild-type enzyme. In contrast, an exonuclease-deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild-type. Conclusions: The hyper-processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence-substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.