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The proofreading exonuclease subunit E of Escherichia coli DNA polymerase III is tethered to the polymerase subunit a via a flexible linker

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posted on 2024-11-14, 15:16 authored by Kiyoshi Ozawa, Slobodan Jergic, Ah-Young Park, Nicholas DixonNicholas Dixon, Gottfried Otting
Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the α-subunit. The ε-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to α via a segment of 57 additional C-terminal residues, and also to θ, whose function is less well defined. The present study shows that θ greatly enhances the solubility of ε during cell-free synthesis. In addition, synthesis of ε in the presence of θ and α resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of ε from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of ε that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of ε is connected to α via a flexible linker peptide comprising over 20 residues. This distinguishes the α : ε complex from other proofreading polymerases, which have a more rigid multidomain structure.

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Citation

Ozawa, K., Jergic, S., Park, A., Dixon, N. E. & Otting, G. (2008). The proofreading exonuclease subunit E of Escherichia coli DNA polymerase III is tethered to the polymerase subunit a via a flexible linker. Nucleic Acids Research, 36 (15), 5074-5082.

Journal title

Nucleic Acids Research

Volume

36

Issue

15

Pagination

5074-5082

Language

English

RIS ID

25699

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