Distal Mutations in the Beta-clamp of DNA Polymerase III* Disrupt DNA Orientation and Affect Exonuclease Activity

DNA polymerases are responsible for the replication and repair of DNA found in all DNA-based organisms. DNA Polymerase III is the main replicative polymerase of E coli and is composed of over 10 proteins. A subset of these proteins (Pol III*) includes the polymerase (alpha), exonuclease (epsilon), clamp (beta) and accessory protein (theta). Mutations of residues in, or around the active site of the catalytic subunits (alpha and epsilon) can have significant impact on catalysis. However, the effects of distal mutations in non-catalytic subunits on the activity of catalytic subunits are less well characterized. Here, we investigate the effects of two Pol III* variants: beta-L82E/L82'E and beta-L82D/L82'D, on the proofreading reaction catalyzed by epsilon. MD simulations reveal major changes in the dynamics of Pol III*, that extend throughout the complex. These changes are mostly induced by a shift in the position of the DNA substrate inside the beta-clamp, although no major structural changes are observed in the protein complex. QM/MM calculations indicate that the beta-L82E/L82'D variant has reduced catalytic proficiency due to highly endoergic reaction energies resulting from structural changes in the active site and differences in the electric field at the active site arising from the protein and substrate. Conversely, the beta-L82E/L82'E variant is predicted to maintain proofreading activity, exhibiting a similar reaction barrier for nucleotide excision compared with the WT system. However, significant differences in the reaction mechanism are obtained due to the changes induced by the mutations on the beta-clamp.