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.
Rancic, S. J., Klassen, K. M., Sawyer, N., Thrall, E. S.
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