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Nucleic Acids Res. 2008 Sep;36(15):5074-82. doi: 10.1093/nar/gkn489. Epub 2008 Jul 28.

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

Author information

1
Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia.

Abstract

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 epsilon-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 epsilon during cell-free synthesis. In addition, synthesis of epsilon 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 epsilon from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of epsilon that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of epsilon is connected to alpha via a flexible linker peptide comprising over 20 residues. This distinguishes the alpha : epsilon complex from other proofreading polymerases, which have a more rigid multidomain structure.

PMID:
18663010
PMCID:
PMC2528190
DOI:
10.1093/nar/gkn489
[Indexed for MEDLINE]
Free PMC Article

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