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.
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.
Yanagihara
Fusamitsu
F
Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Yoshida
Shohei
S
Sugaya
Yutaka
Y
Maki
Hisaji
H
D019943:Amino Acid Substitution; D001665:Binding Sites; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D011993:Recombinant Fusion Proteins; D004258:DNA Polymerase III