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