Stoichiometry of Active DNA Replication Machinery Within Living Escherichia Coli Cells

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One of our missions is to explore the biology of the bacterial chromosome at single molecule resolution in live cells in close to real-time. Our focus is to analyze the assembly, action and disassembly of the molecular machine that replicates DNA [the replisome] in living E. coli cells and then use targeted protein ablation, using specific degron tags, to remove individual replisome components. The composition and architecture of the replisome in actively replicating cells is unknown, despite extensive characterization of the major replication components and their use to reconstitute bacterial and bacteriophage replication in vitro. By using a novel fluorescence microscopy technique [‘slimfield’microscopy], with single molecule sensitivity, simultaneous multi-color capture and 3 ms temporal resolution, on living cells expressing fluorescent derivatives of eight replisome components from their endogenous promoters, we show that active Escherichia coli replisomes in vivo contain one clamp loader complex associated with three molecules each of polymerase and DnaX. Comparison of the spatial distribution of the three sliding clamps and polymerases, shows that ∼3/4 of replisomes have only two elongating polymerases associated with a sliding clamp at any one time. HolC and HolD, which heterodimerize to link the clamp loader to Ssb, are most frequently present as 2-5 molecules per replisome. Turnover of Ssb, which coats the single-stranded lagging DNA template, is ∼2-fold faster in replicating cells than in replication-inhibited cells, with most active replisomes being associated with 5-11 Ssb tetramers. Replication-inhibited replisomes have a ∼2-fold increase in Ssb associated with each fork.