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

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