The role of the DnaN sliding clamp interaction in DNA replication and mutagenesis in B. subtilis

Ring-shaped sliding clamp proteins are essential components of the replication machinery, the replisome, across all domains of life. In bacteria, DNA polymerases bind the sliding clamp, DnaN, through conserved short peptide sequences called clamp-binding motifs. Clamp binding increases the processivity and rate of DNA synthesis and is generally required for polymerase activity. The gram-negative bacterium Escherichia coli, which utilizes one replicative polymerase, has served as the model species for bacterial DNA replication. However, many other bacteria have two essential replicative polymerases, such as PolC and DnaE in the gram-positive species Bacillus subtilis. In the current model, PolC performs the bulk of DNA synthesis whereas DnaE, which is error-prone, only synthesizes short stretches of DNA on the lagging strand. The role of the clamp interaction in coordinating polymerase activity in B. subtilis is unknown. We investigated this question by combining in vivo single-molecule fluorescence microscopy with biochemical and microbiological assays. We found that PolC-DnaN binding is essential for replication, although weakening the interaction is tolerated with only minimal effects. In contrast, the DnaE-DnaN interaction is dispensable for replication. Altering the clamp-binding strength produces subtle effects on the cellular localization and dynamics of DnaE but has a substantial effect on mutagenesis. Our results support a model where DnaE generally acts distributively during replication but can be stabilized on the DNA template by clamp binding. Although PolC must bind the clamp during replication, our findings suggest that other interactions may play a role in stabilizing PolC at the replication fork. This study provides new insight into the coordination of multiple replicative polymerases in bacteria as well as the role of the clamp in polymerase trafficking more broadly.