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Abstract 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 bacteriumEscherichia coli, which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE inBacillus 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. 

Abstract Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.