A New Role for the Proofreader in Bacterial DNA Replication

Abstract

The DNA polymerase III holoenzyme (Pol III HE), the E. coli chromosomal replicase, is made up of 2 (or 3) αεθ core polymerases, 3 dimeric β sliding clamps, and the 7‐subunit clamp loader that loads the clamp onto primer‐templates. When bound to the clamp, the Pol III core becomes fast (~750 nt/s) and processive (>50 kb) due to the α‐β interaction. The ε subunit is the proofreading 3′→5′ exonuclease that interacts with α and greatly increases the fidelity of Pol III HE. Genetic studies 20 years ago suggested that ε has an essential role in replication beyond its contribution to fidelity, and we have previously also shown that it is attached to α via a flexible linker that permits it to sample a relatively large space around α in the replicase. Here, we present new functional assays (Pol III strand displacement and primer extension under difficult conditions) that expose the additional role of ε in DNA replication. Bioinformatics indicated it may be mediated by a new interaction with the β clamp. We further utilized SPR and ESI‐MS to obtain direct evidence for a physical interaction between ε and β both in isolation and within the Pol III‐clamp complex. Single molecule DNA replication assays revealed the role of the relatively weak yet important ε‐β contact in stabilization of the polymerase on the DNA template.