The B. subtilis replicative polymerases bind the sliding clamp with different strengths to tune their activity in DNA replication

AbstractRing-shaped sliding clamp proteins are essential components of the replication machinery across all domains of life. DNA polymerases bind the clamp, increasing the processivity and rate of DNA synthesis. The current understanding of bacterial clamp-polymerase interactions was elucidated in Escherichia coli , which has one replicative polymerase. 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, primarily on the lagging strand. Whether the clamp, DnaN, 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, although weakening the interaction is tolerated with 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 leads to increased 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 how clamp binding coordinates multiple replicative polymerases in bacteria.Graphical AbstractGraphical Abstract