Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli.
Sugaya, Yutaka 1; Ihara, Keiichi 2; Masuda, Yuji 1,a; Ohtsubo, Eiichi 2; Maki, Hisaji 1,2,*
[Article] Genes to Cells. 7(4):385-399, April 2002.

(Format: HTML, PDF)

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 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.

(C) 2002 Blackwell Science Ltd.