The dnaE173 mutator mutation confers on the alpha subunit of Escherichia coli DNA polymerase III a capacity for highly processive DNA synthesis and stable binding to primer/template DNA

Genes Genet Syst. 2007 Aug;82(4):273-80. doi: 10.1266/ggs.82.273.

Abstract

The strong mutator mutation dnaE173 which causes an amino-acid substitution in the alpha subunit of DNA polymerase III is unique in its ability to induce sequence-substitution mutations. We showed previously that multiple biochemical properties of DNA polymerase III holoenzyme of Escherichia coli are simultaneously affected by the dnaE173 mutation. These effects include a severely reduced proofreading capacity, an increased resistance to replication-pausing on the template DNA, a capability to readily promote strand-displacement DNA synthesis, a reduced rate of DNA chain elongation, and an ability to catalyze highly processive DNA synthesis in the absence of the beta-clamp subunit. Here we show that, in contrast to distributive DNA synthesis exhibited by wild-type alpha subunit, the dnaE173 mutant form of alpha subunit catalyzes highly processive DNA chain elongation without the aid of the beta-clamp. More surprisingly, the dnaE173 alpha subunit appeared to form a stable complex with primer/template DNA, while no such affinity was detected with wild-type alpha subunit. We consider that the highly increased affinity of alpha subunit for primer/template DNA is the basis for the pleiotropic effects of the dnaE173 mutation on DNA polymerase III, and provides a clue to the molecular mechanisms underlying sequence substitution mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution*
  • Binding Sites
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Primers
  • DNA Replication
  • DNA, Bacterial / biosynthesis*
  • DNA-Binding Proteins / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / metabolism*
  • Holoenzymes / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Recombinant Fusion Proteins
  • Templates, Genetic

Substances

  • DNA Primers
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Holoenzymes
  • Recombinant Fusion Proteins
  • DNA Polymerase III