Sawyer, N. | Search bioRxiv

bioRxiv2026

Characterizing the Molecular Determinants of Clamp Binding in B. subtilis

In bacteria, the ring-shaped sliding clamp, DnaN, is an essential component of the replication machinery. The clamp encircles the parental DNA strand during replication and binds DNA polymerases and other replication and repair proteins, helping to tether them at their site of action on the DNA strand. These binding partners interact with the clamp via short pentapeptide or hexapeptide sequences known as clamp-binding motifs (CBMs). Although conserved CBM sequences have been identified across different bacterial species, most studies of clamp binding have been performed in the model gram-negative bacterium Escherichia coli and less is known about clamp binding in other bacterial species. In this study, we investigate clamp binding in the model gram-positive bacterium Bacillus subtilis. We use fluorescence polarization binding assays to quantify binding of a range of CBM peptides to the clamps of both E. coli and B. subtilis. We identify similarities in clamp binding between the two species, including similar importance of different amino acids within the conserved pentapeptide motif. However, our results also reveal differences in clamp binding between the two species. Most notably, we find that, although pentapeptide CBMs bind the E. coli and B. subtilis clamps with similar affinity, hexapeptide CBMs bind an order of magnitude more weakly to the B. subtilis clamp. Our results provide new insight into clamp binding in bacteria and point to possible species-specific differences in this essential interaction.

biochemistry↗

bioRxiv2025

The B. subtilis replicative polymerases bind the sliding clamp with different strengths to tune replication processivity and fidelity

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.

biochemistry↗