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Hyper‐processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli
Author(s) -
Sugaya Yutaka,
Ihara Keiichi,
Masuda Yuji,
Ohtsubo Eiichi,
Maki Hisaji
Publication year - 2002
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2002.00527.x
Subject(s) - biology , processivity , dna polymerase , dna clamp , exonuclease , proofreading , dna polymerase ii , polymerase , dna polymerase delta , protein subunit , dna polymerase i , dna replication , microbiology and biotechnology , biochemistry , dna , mutant , polymerase chain reaction , gene , reverse transcriptase
Background: A strong mutator mutation, dnaE173 , leads to a Glu 612 → Lys amino acid change in the α 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 ɛ subunit. The dnaE173 mutator is unique in its ability to induce sequence‐substitution mutations, suggesting that an unknown function of the α subunit is hampered by the dnaE173 mutation. Results: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the β clamp subunit) and the β 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 β‐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.