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

被引:20
作者
Sugaya, Y
Ihara, K
Masuda, Y
Ohtsubo, E
Maki, H [1 ]
机构
[1] Nara Inst Sci & Technol, Grad Sch Biol Sci, Dept Mol Biol, Nara 6300101, Japan
[2] Univ Tokyo, Inst Mol & Cellular Biol, Bunkyo Ku, Tokyo 1130032, Japan
关键词
D O I
10.1046/j.1365-2443.2002.00527.x
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Background: A strong mutator mutation, dnaE173, leads to a Glu(612) --> Lys amino acid change in the a 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.
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页码:385 / 399
页数:15
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