Format

Send to

Choose Destination
Genes Cells. 2002 Apr;7(4):385-99.

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

Author information

1
Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.

Abstract

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.

[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center