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 current understanding of clamp-polymerase interactions was elucidated in the model bacterium Escherichia coli , which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE in Bacillus subtilis . PolC performs the bulk of DNA synthesis whereas the error-p... More
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 current understanding of clamp-polymerase interactions was elucidated in the model bacterium Escherichia coli , which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE in Bacillus subtilis . PolC performs the bulk of DNA synthesis whereas the error-prone DnaE only synthesizes short stretches of DNA on the lagging strand. How the clamp interacts with the two polymerases and coordinates their activity 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 of DnaE produces only subtle effects on DnaE cellular localization and dynamics, but it has a substantial impact on mutagenesis. Our results support a model in which DnaE acts distributively during replication but can be stabilized on the DNA template by clamp binding. This study provides new insights into the coordination of multiple replicative polymerases in bacteria and the role of the clamp in polymerase processivity, fidelity, and exchange.
