Long-Range Destabilizing Effects of Mutations at the

The bacterial processivity factor, the β sliding clamp, endows Pol III DNA polymerase α with efficiency and processivity during DNA replication by tethering α to DNA. The dimeric β clamp is loaded onto primer:template DNA junctions by a five-subunit clamp loader complex. We previously showed that tuning the stability of the β dimer interface affected clamp opening dynamics and function. The L82D clamp mutation destabilized the dimer interface due to altered hydrophobic interactions and β L82D exhibited lower thermostability, a change in dimerization state, and a decrease in loading activity. Molecular modeling revealed a rearrangement of the local hydrogen-bond network near L82D in Domain 1, leading to new, non-native interactions involving residues Q265 and R269 in Domain 3 of the partner protomer. We hypothesize that altering this newly formed network in the context of the β L82D variant may stabilize the dimer interface and rescue clamp activity. To test these hypotheses, Q265 and R269 were mutated in WT β and in the L82D variant. The β R269 residue is essential for maintaining a stable dimer interface, functional oligomeric state, and efficient DNA loading, while Q265 is not, as Q265A is tolerated. Disrupting native interactions of R269 with the formation of an alternate bonding network in L82D seems to be the primary driver for the lower thermostability, altered dimerization state, and loss of clamp activity previously observed in β L82D. Our observations show that a complex balance of noncovalent interactions contributes to the stability of the β clamp dimer interface.