Investigating the Dynamics of the β2 Sliding Clamp in Escherichia Coli at the Single-Cell Level Utilizing Single-Molecule Fluorescence Microscopy

Abstract

An E.coli cell contains a single circular chromosomal DNA, and recent studies have shown that this chromosome is duplicated in an independent bidirectional manner by two individual replisomes prior to cell division. Here, we investigate the dynamics of the replisome complex, specifically the β2 sliding clamp, in living cells. We use a combination of microfluidics and single-molecule fluorescence microscopy to track the replisomes in individual E.coli cells, whereby the native β2 clamp proteins are labeled with a fluorophore (mYpet) by chromosomal fusion. To carry out these experiments, we have reliably quantified the fluorescence signal generated by the β-mYpet fusion inside a growing cell by comparing numerous in vitro and in vivo calibration standards. Additionally, our use of microfluidics has increased the yield of the measurement with respect to the number of analyzed cells and the measurement time, as well as facilitated automated analysis using customized MATLAB scripts. The results of our experiments clearly demonstrate that the β2 proteins are temporally and spatially dynamic during a cell cycle: following initiation, there is a gradual increase in the number of β2 clamps bound to the DNA until a steady state is reached. This steady state, which surprisingly involves approximately 50% of the β2 proteins inside the cell, is maintained for most of the cell cycle until a gradual decrease is observed around 15 minutes before termination of replication. We conclude that there is a clear accumulation of β2 clamps along the two replication forks, and we propose a model that the β2 clamps are left behind on the lagging strand upon synthesis of each Okazaki fragment as the replication fork progresses.