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

Genes Cells. 2002 Apr;7(4):385-99. doi: 10.1046/j.1365-2443.2002.00527.x.


Background: A strong mutator mutation, dnaE173, leads to a Glu612 --> Lys amino acid change in the alpha 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 epsilon subunit. The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the alpha subunit is hampered by the dnaE173 mutation.

Results: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the beta clamp subunit) and the beta 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 beta-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Replication / physiology*
  • DNA, Bacterial / biosynthesis*
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Holoenzymes / metabolism
  • Mutation
  • Potassium Chloride / metabolism
  • Silver Staining
  • Thymidine Monophosphate / metabolism


  • DNA, Bacterial
  • DNA-Binding Proteins
  • Holoenzymes
  • Thymidine Monophosphate
  • Potassium Chloride
  • Adenosine Triphosphate
  • beta subunit, DNA polymerase III
  • DNA Polymerase III