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Nucleic Acids Research Advance Access originally published online on July 28, 2008
Nucleic Acids Research 2008 36(15):5074-5082; doi:10.1093/nar/gkn489
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Nucleic Acids Research, 2008, Vol. 36, No. 15 5074-5082
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Structural Biology

The proofreading exonuclease subunit {varepsilon} of Escherichia coli DNA polymerase III is tethered to the polymerase subunit {alpha} via a flexible linker

Kiyoshi Ozawa1, Slobodan Jergic1,2, Ah Young Park1, Nicholas E. Dixon1,2 and Gottfried Otting1,*

1Research School of Chemistry, Australian National University, Canberra ACT 0200 and 2School of Chemistry, University of Wollongong, NSW 2522, Australia

*To whom correspondence should be addressed. Tel: +61 2 61256507; Fax: +61 2 61250750; Email: go{at}rsc.anu.edu.au

Received June 5, 2008. Revised July 14, 2008. Accepted July 16, 2008.

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


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