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