Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli.
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.
Sugaya
Yutaka
Y
Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Ihara
Keiichi
K
Masuda
Yuji
Y
Ohtsubo
Eiichi
E
Maki
Hisaji
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D020035:Holoenzymes / Q000378:metabolism; D009154:Mutation; D011189:Potassium Chloride / Q000378:metabolism; D016622:Silver Staining; D013938:Thymidine Monophosphate / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D013938:Thymidine Monophosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III