Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells

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 beta(2)- sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both beta(2)-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with singlemolecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of beta(2)-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous beta(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.