Slow unloading leads to DNA-bound β ₂-sliding clamp accumulation in live Escherichia coli cells

Abstract

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β ₂-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β ₂-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β ₂-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β ₂-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.

Abstract

DNA replication is accomplished by the replisome, a multi-protein complex that comprises the sliding clamp. Here, Moolman et al. present quantitative and dynamic measurements of the number of β ₂-sliding clamps at the single-cell level in live E. coli cells to shed light on key aspects of DNA replication.